Behavioral plasticity.

Behavioral plasticity allows animals to modulate their behavior based on experience and environmental conditions. Caenorhabditis elegans exhibits experience-dependent changes in its behavioral responses to various modalities of sensory cues, including odorants, salts, temperature, and mechanical stimulations. Most of these forms of behavioral plasticity, such as adaptation, habituation, associative learning, and imprinting, are shared with other animals. The C. elegans nervous system is considerably tractable for experimental studies-its function can be characterized and manipulated with molecular genetic methods, its activity can be visualized and analyzed with imaging approaches, and the connectivity of its relatively small number of neurons are well described. Therefore, C. elegans provides an opportunity to study molecular, neuronal, and circuit mechanisms underlying behavioral plasticity that are either conserved in other animals or unique to this species. These findings reveal insights into how the nervous system interacts with the environmental cues to generate behavioral changes with adaptive values.

Uncommon opsin's retinal isomer is involved in mammalian sperm thermotaxis.

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts-tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

Development of a thermotaxis and rheotaxis microfluidic device for motile spermatozoa sorting.

Male infertility is a pervasive global reproductive challenge, primarily attributed to a decline in semen quality. Addressing this concern, there has been a growing focus on spermatozoa sorting in assisted reproductive technology. This study introduces a groundbreaking development in the form of a thermotaxis and rheotaxis microfluidic (TRMC) device designed for efficient motile spermatozoa sorting within a short 15-min timeframe. The TRMC device mimics the natural sperm sorting mechanism of the oviduct, selecting spermatozoa with superior motility and DNA integrity. The experimental outcomes demonstrate a remarkable enhancement in the percentage of progressive spermatozoa following sorting, soaring from 3.90% to an impressive 96.11% when subjected to a temperature decrease from 38 °C to 35 °C. Notably, sperm motility exhibited a substantial 69% improvement. The TRMC device exhibited a commendable recovery rate of 60.93%, surpassing current clinical requirements. Furthermore, the sorted spermatozoa displayed a notable reduction in the DNA fragmentation index to 6.94%, signifying a substantial 90% enhancement in DNA integrity. This remarkable advancement positions the TRMC device as highly suitable for applications in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offering a promising solution to male infertility challenges.

A hyperpolarizing neuron recruits undocked innexin hemichannels to transmit neural information in Caenorhabditis elegans.

While depolarization of the neuronal membrane is known to evoke the neurotransmitter release from synaptic vesicles, hyperpolarization is regarded as a resting state of chemical neurotransmission. Here, we report that hyperpolarizing neurons can actively signal neural information by employing undocked hemichannels. We show that UNC-7, a member of the innexin family in Caenorhabditis elegans, functions as a hemichannel in thermosensory neurons and transmits temperature information from the thermosensory neurons to their postsynaptic interneurons. By monitoring neural activities in freely behaving animals, we find that hyperpolarizing thermosensory neurons inhibit the activity of the interneurons and that UNC-7 hemichannels regulate this process. UNC-7 is required to control thermotaxis behavior and functions independently of synaptic vesicle exocytosis. Our findings suggest that innexin hemichannels mediate neurotransmission from hyperpolarizing neurons in a manner that is distinct from the synaptic transmission, expanding the way of neural circuitry operations.

Temperature-dependent augmentation of ciliary motility by the TRP2 channel in Chlamydomonas reinhardtii.

Temperature is a critical factor for living organisms. Many microorganisms migrate toward preferable temperatures, and this behavior is called thermotaxis. In this study, the molecular and physiological bases for thermotaxis are examined in Chlamydomonas reinhardtii. A mutant with knockout of a transient receptor potential (TRP) channel, trp2-3, showed defective thermotaxis. The swimming velocity and ciliary beat frequency of wild-type Chlamydomonas increase with temperature; however, this temperature-dependent enhancement of motility was almost absent in the trp2-3 mutant. Wild-type Chlamydomonas showed negative thermotaxis, but mutants deficient in the outer or inner dynein arm showed positive thermotaxis and a defect in temperature-dependent increase in swimming velocity, suggesting involvement of both dynein arms in thermotaxis.

Microfluidics: The future of sperm selection in assisted reproduction.

BACKGROUND: Obtaining functional sperm cells is the first step to treat infertility. With the ever-increasing trend in male infertility, clinicians require access to effective solutions that are able to single out the most viable spermatozoa, which would max out the chance for a successful pregnancy. The new generation techniques for sperm selection involve microfluidics, which offers laminar flow and low Reynolds number within the platforms can provide unprecedented opportunities for sperm selection. Previous studies showed that microfluidic platforms can provide a novel approach to this challenge and since then researchers across the globe have attacked this problem from multiple angles. OBJECTIVE: In this review, we seek to provide a much-needed bridge between the technical and medical aspects of microfluidic sperm selection. Here, we provide an up-to-date list on microfluidic sperm selection procedures and its application in assisted reproductive technology laboratories. SEARCH METHOD: A literature search was performed in Web of Science, PubMed, and Scopus to select papers reporting microfluidic sperm selection using the keywords: microfluidic sperm selection, self-motility, non-motile sperm selection, boundary following, rheotaxis, chemotaxis, and thermotaxis. Papers published before March 31, 2023 were selected. OUTCOMES: Our results show that most studies have used motility-based properties for sperm selection. However, microfluidic platforms are ripe for making use of other properties such as chemotaxis and especially rheotaxis. We have identified that low throughput is one of the major hurdles to current microfluidic sperm selection chips, which can be solved via parallelization. CONCLUSION: Future work needs to be performed on numerical simulation of the microfluidics chip prior to fabrication as well as relevant clinical assessment after the selection procedure. This would require a close collaboration and understanding among engineers, biologists, and medical professionals. It is interesting that in spite of two decades of microfluidics sperm selection, numerical simulation and clinical studies are lagging behind. It is expected that microfluidic sperm selection platforms will play a major role in the development of fully integrated start-to-finish assisted reproductive technology systems.

Temperature sensitivity and temperature response across development in the Drosophila larva.

The surrounding thermal environment is highly important for the survival and fitness of animals, and as a result most exhibit behavioral and neural responses to temperature changes. We study signals generated by thermosensory neurons in Drosophila larvae and also the physical and sensory effects of temperature variation on locomotion and navigation. In particular we characterize how sensory neuronal and behavioral responses to temperature variation both change across the development of the larva. Looking at a wide range of non-nociceptive isotropic thermal environments, we characterize the dependence of speed, turning rate, and other behavioral components on temperature, distinguishing the physical effects of temperature from behavior changes based on sensory processing. We also characterize the strategies larvae use to modulate individual behavioral components to produce directed navigation along thermal gradients, and how these strategies change during physical development. Simulations based on modified random walks show where thermotaxis in each developmental stage fits into the larger context of possible navigation strategies. We also investigate cool sensing neurons in the larva's dorsal organ ganglion, characterizing neural response to sine-wave modulation of temperature while performing single-cell-resolution 3D imaging. We determine the sensitivity of these neurons, which produce signals in response to extremely small temperature changes. Combining thermotaxis results with neurophysiology data, we observe, across development, sensitivity to temperature change as low as a few thousandths of a °C per second, or a few hundredths of a °C in absolute temperature change.

Involvement of cyclic nucleotide-gated channels in soybean cyst nematode chemotaxis and thermotaxis.

The soybean cyst nematode (SCN) is one of the most damaging pests affecting soybean production. SCN displays important host recognition behaviors, such as hatching and infection, by recognizing several compounds produced by the host. Therefore, controlling SCN behaviors such as chemotaxis and thermotaxis is an attractive pest control strategy. In this study, we found that cyclic nucleotide-gated channels (CNG channels) regulate SCN chemotaxis and thermotaxis and Hg-tax-2, a gene encoding a CNG channel, is an important regulator of SCN behavior. Gene silencing of Hg-tax-2 and treatment with a CNG channel inhibitor reduced the attraction of second-stage juveniles to nitrate, an attractant with a different recognition mechanism from the host-derived chemoattractant(s), and to host soybean roots, as well as their avoidance behavior toward high temperatures. Co-treatment of ds Hg-tax-2 with the CNG channel inhibitor indicated that Hg-tax-2 is a major regulator of SCN chemotaxis and thermotaxis. These results suggest new avenues for research on control of SCN.

Thermotaxis in an apolar, non-neuronal animal.

Neuronal circuits are hallmarks of complex decision-making processes in the animal world. How animals without neurons process information and respond to environmental cues promises a new window into studying precursors of neuronal control and origin of the nervous system as we know it today. Robust decision making in animals, such as in chemotaxis or thermotaxis, often requires internal symmetry breaking (such as anterior-posterior (AP) axis) provided naturally by a given body plan of an animal. Here we report the discovery of robust thermotaxis behaviour in Trichoplax adhaerens, an early-divergent, enigmatic animal with no anterior-posterior symmetry breaking (apolar) and no known neurons or muscles. We present a quantitative and robust behavioural response assay in Placozoa, which presents an apolar flat geometry. By exposing T. adhaerens to a thermal gradient under a long-term imaging set-up, we observe robust thermotaxis that occurs over timescale of hours, independent of any circadian rhythms. We quantify that T. adhaerens can detect thermal gradients of at least 0.1°C cm-1. Positive thermotaxis is observed for a range of baseline temperatures from 17°C to 22.5°C, and distributions of momentary speeds for both thermotaxis and control conditions are well described by single exponential fits. Interestingly, the organism does not maintain a fixed orientation while performing thermotaxis. Using natural diversity in size of adult organisms (100 µm to a few millimetres), we find no apparent size-dependence in thermotaxis behaviour across an order of magnitude of organism size. Several transient receptor potential (TRP) family homologues have been previously reported to be conserved in metazoans, including in T. adhaerens. We discover naringenin, a known TRPM3 antagonist, inhibits thermotaxis in T. adhaerens. The discovery of robust thermotaxis in T. adhaerens provides a tractable handle to interrogate information processing in a brainless animal. Understanding how divergent marine animals process thermal cues is also critical due to rapid temperature rise in our oceans.

Identification of a spontaneously arising variant affecting thermotaxis behavior in a recombinant inbred Caenorhabditis elegans line.

Analyses of the contributions of genetic variants in wild strains to phenotypic differences have led to a more complete description of the pathways underlying cellular functions. Causal loci are typically identified via interbreeding of strains with distinct phenotypes in order to establish recombinant inbred lines (RILs). Since the generation of RILs requires growth for multiple generations, their genomes may contain not only different combinations of parental alleles but also genetic changes that arose de novo during the establishment of these lines. Here, we report that in the course of generating RILs between Caenorhabditis elegans strains that exhibit distinct thermotaxis behavioral phenotypes, we identified spontaneously arising variants in the ttx-1 locus. ttx-1 encodes the terminal selector factor for the AFD thermosensory neurons, and loss-of-function mutations in ttx-1 abolish thermotaxis behaviors. The identified genetic changes in ttx-1 in the RIL are predicted to decrease ttx-1 function in part via specifically affecting a subset of AFD-expressed ttx-1 isoforms. Introduction of the relevant missense mutation in the laboratory C. elegans strain via gene editing recapitulates the thermotaxis behavioral defects of the RIL. Our results suggest that spontaneously occurring genomic changes in RILs may complicate identification of loci contributing to phenotypic variation, but that these mutations may nevertheless lead to the identification of important causal molecules and mechanisms.

Human scent guides mosquito thermotaxis and host selection under naturalistic conditions.

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sensory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiologically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An. gambiae prefers to land on arrayed visual targets warmed to human skin temperature during the nighttime when they are baited with carbon dioxide (CO2) emissions reflective of a large human over background air, body odor from one human over CO2, and the scent of one sleeping human over another. Applying integrative whole body volatilomics to multiple humans tested simultaneously in competition in a six-choice assay, we reveal high attractiveness is associated with whole body odor profiles from humans with increased relative abundances of the volatile carboxylic acids butyric acid, isobutryic acid, and isovaleric acid, and the skin microbe-generated methyl ketone acetoin. Conversely, those least preferred had whole body odor that was depleted of carboxylic acids among other compounds and enriched with the monoterpenoid eucalyptol. Across expansive spatial scales, heated targets without CO2 or whole body odor were minimally or not attractive at all to An. gambiae. These results indicate that human scent acts critically to guide thermotaxis and host selection by this prolific malaria vector as it navigates towards humans, yielding intrinsic heterogeneity in human biting risk.

Molecular encoding of stimulus features in a single sensory neuron type enables neuronal and behavioral plasticity.

Neurons modify their transcriptomes in response to an animal's experience. How specific experiences are transduced to modulate gene expression and precisely tune neuronal functions are not fully defined. Here, we describe the molecular profile of a thermosensory neuron pair in C. elegans experiencing different temperature stimuli. We find that distinct salient features of the temperature stimulus, including its duration, magnitude of change, and absolute value, are encoded in the gene expression program in this single neuron type, and we identify a novel transmembrane protein and a transcription factor whose specific transcriptional dynamics are essential to drive neuronal, behavioral, and developmental plasticity. Expression changes are driven by broadly expressed activity-dependent transcription factors and corresponding cis-regulatory elements that nevertheless direct neuron- and stimulus-specific gene expression programs. Our results indicate that coupling of defined stimulus characteristics to the gene regulatory logic in individual specialized neuron types can customize neuronal properties to drive precise behavioral adaptation.

Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome.

BACKGROUND: Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. METHODS: We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. RESULTS: The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05). CONCLUSION: Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates.

Lab-on-chip (LoC) application for quality sperm selection: An undelivered promise?

Quality sperm selection is essential to ensure the effectiveness of assisted reproductive techniques (ART). However, the methods employed for sperm selection in ART often yield suboptimal outcomes, contributing to lower success rates. In recent years, microfluidic devices have emerged as a promising avenue for investigating the natural swimming behavior of spermatozoa and developing innovative approaches for quality sperm selection. Despite their potential, the commercial translation of microfluidic-based technologies has remained limited. This comprehensive review aims to critically evaluate the inherent potential of lab-on-chip technology in unraveling sophisticated mechanisms encompassing rheotaxis, thermotaxis, and chemotaxis. By reviewing the current state-of-the-art associated with microfluidic engineering and the swimming of spermatozoa, the goal is to shed light on the multifaceted factors that have impeded the broader commercialization of these cutting-edge technologies and recommend a commercial that can surmount the prevailing constraints. Furthermore, this scholarly exploration seeks to enlighten and actively engage reproductive clinicians in the profound potential and implications of microfluidic methodologies within the context of human infertility.

Cool and warm ionotropic receptors control multiple thermotaxes in Drosophila larvae.

Animals are continuously confronted with different rates of temperature variation. The mechanism underlying how temperature-sensing systems detect and respond to fast and slow temperature changes is not fully understood in fly larvae. Here, we applied two-choice behavioral assays to mimic fast temperature variations and a gradient assay to model slow temperature changes. Previous research indicates that Rhodopsin 1 (Rh1) and its phospholipase C (PLC) cascade regulate fast and slow temperature responses. We focused on the ionotropic receptors (IRs) expressed in dorsal organ ganglions (DOG), in which dorsal organ cool-activated cells (DOCCs) and warm-activated cells (DOWCs) rely on IR-formed cool and warm receptors to respond to temperature changes. In two-choice assays, both cool and warm IRs are sufficient for selecting 18°C between 18°C and 25°C but neither function in cool preferences between 25°C and 32°C. The Rh1 pathway, on the other hand, contributes to choosing preferred temperatures in both assays. In a gradient assay, cool and warm IR receptors exert opposite effects to guide animals to ∼25°C. Cool IRs drive animals to avoid cool temperatures, whereas warm IRs guide them to leave warm regions. The Rh1 cascade and warm IRs may function in the same pathway to drive warm avoidance in gradient assays. Moreover, IR92a is not expressed in temperature-responsive neurons but regulates the activation of DOWCs and the deactivation of DOCCs. Together with previous studies, we conclude that multiple thermosensory systems, in various collaborative ways, help larvae to make their optimal choices in response to different rates of temperature change.

Microfluidic devices employing chemo- and thermotaxis for sperm selection can improve sperm parameters and function in patients with high DNA fragmentation.

Conventional sperm processing uses centrifugation has a negative effect on sperm parameters and DNA integrity. We designed and fabricated a novel microfluid device based on chemotaxis and thermotaxis, and compared it with the swim-up method. Twenty normal samples with high DNA fragmentation were included. Each sample was divided into four groups: Group 1, control, Group 2: sperm selection by thermotaxis, Group 3: sperm selection by chemotaxis, and Group 4: sperm selection with thermotaxis and chemotaxis. We used cumulus cells in a microfluid device to create chemotaxis, and, two warm stages to form a temperature gradient for thermotaxis. The spermatozoa were assessed based on the concentration, motility, and fine morphology using Motile Sperm Organelle Morphology Examination, mitochondrial membrane potential (MMP), acrosome reaction (AR), and sperm DNA fragmentation. Concentration (22.40 ± 5.39 vs. 66.50 ± 19.21; p < 0.001) and DNA fragmentation (12.30 ± 3.96% vs. 17.95 ± 2.89%; p < 0.001) after selection in the chemotaxis and thermotaxis microfluid device were significantly lower than control group. The progressive motility (93.75 ± 4.39% vs. 75.55 ± 5.86%, p < 0.001), normal morphology (15.45 ± 2.50% vs. 10.35 ± 3.36, p < 0.001), MMP (97.65 ± 1.81% vs. 94 ± 3.89%, p = 0.02), and AR status (79.20 ± 5.28% vs. 31.20 ± 5.24%, p < 0.001) in the chemotaxis and thermotaxis microfluid device were significantly increased compared to control group. According to these findings, spermatozoa that have penetrated the cumulus oophorus have better morphology and motility, as well as acrosome reactivity and DNA integrity.

Thermotaxic diel vertical migration of the harmful dinoflagellate Cochlodinium (Margalefidinium) polykrikoides: Combined field and laboratory studies.

The harmful dinoflagellate Cochlodinium polykrikoides, a species that causes mass mortality of farmed fish, uses diel vertical migration (DVM) as an ecological strategy. In summer 2018, a bloom of C. polykrikoides occurred on the southern coast of Korea when the surface water temperature exceeded 29 °C, as a result of a marine heatwave. To understand the effect of high temperature conditions on the DVM of C. polykrikoides, vertical profiles of environmental variables and the occurrence of the dinoflagellate were investigated through a 48 h field survey. In addition, a thermally stratified environment (6-12 °C difference between the surface and bottom layers) was established in a laboratory study to investigate the effect of temperature difference between water layers on the DVM of C. polykrikoides. In the field, most of the C. polykrikoides population was at a depth of 3-6 m during the day, where the water temperature was significantly lower (p < 0.01; Chi square = 57.98; Kruskal-Wallis test) than in the surface layer (0 m), and only the water temperature at 0 m was not correlated with weighted mean depth of C. polykrikoides, suggesting the usage of DVM to avoid high temperature stress. According to our field and laboratory results, there was a trend of greater DVM velocity by thermotaxis when moving from "unfavorable" water temperature (30 °C hot and 12 °C cold) to "favorable" water temperature for growth (optimal 24 °C) of C. polykrikoides. Our findings suggest that thermotaxic DVM is an important ecological strategy used by C. polykrikoides to optimize environmental conditions for growth through vertical positioning and changing migration velocity.

Genetic screens identified dual roles of microtubule-associated serine threonine kinase and CREB within a single thermosensory neuron in the regulation of Caenorhabditis elegans thermotaxis behavior.

Animals integrate sensory stimuli presented at the past and present, assess the changes in their surroundings and navigate themselves toward preferred environment. Identifying the neural mechanisms of such sensory integration is pivotal to understand how the nervous system generates perception and behavior. Previous studies on thermotaxis behavior of Caenorhabditis elegans suggested that a single thermosensory neuron AFD plays an important role in integrating the past and present temperature information and is essential for the neural computation that drives the animal toward the preferred temperature region. However, the molecular mechanisms by which AFD executes this neural function remained elusive. Here we report multiple forward genetic screens to identify genes required for thermotaxis. We reveal that kin-4, which encodes the C. elegans homolog of microtubule-associated serine threonine kinase, plays dual roles in thermotaxis and can promote both cryophilic and thermophilic drives. We also uncover that a thermophilic defect of mutants for mec-2, which encodes a C. elegans homolog of stomatin, can be suppressed by a loss-of-function mutation in the gene crh-1, encoding a C. elegans homolog CREB transcription factor. Expression of crh-1 in AFD restored the crh-1-dependent suppression of the mec-2 thermotaxis phenotype, indicating that crh-1 can function in AFD to regulate thermotaxis. Calcium imaging analysis from freely moving animals suggest that mec-2 and crh-1 regulate the neuronal activity of the AIY interneuron, a postsynaptic partner of the AFD neuron. Our results suggest that a stomatin family protein can control the dynamics of neural circuitry through the CREB-dependent transcriptional regulation within a sensory neuron.

A miniaturized electrothermal array for rapid analysis of temperature preference behaviors in ecology and ecotoxicology.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants.

Sensory-Motor Perturbations in Larval Zebrafish (Danio rerio) Induced by Exposure to Low Levels of Neuroactive Micropollutants during Development.

Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.