Invited Review Beyond parasitic convergence: unravelling the evolution of the organellar genomes in holoparasites.

BACKGROUND: The molecular evolution of organellar genomes in angiosperms has been studied extensively, with some lineages, such as parasitic ones, displaying unique characteristics. Parasitism has emerged 12 times independently in angiosperm evolution. Holoparasitism is the most severe form of parasitism, and is found in ~10 % of parasitic angiosperms. Although a few holoparasitic species have been examined at the molecular level, most reports involve plastomes instead of mitogenomes. Parasitic plants establish vascular connections with their hosts through haustoria to obtain water and nutrients, which facilitates the exchange of genetic information, making them more susceptible to horizontal gene transfer (HGT). HGT is more prevalent in the mitochondria than in the chloroplast or nuclear compartments. SCOPE: This review summarizes current knowledge on the plastid and mitochondrial genomes of holoparasitic angiosperms, compares the genomic features across the different lineages, and discusses their convergent evolutionary trajectories and distinctive features. We focused on Balanophoraceae (Santalales), which exhibits extraordinary traits in both their organelles. CONCLUSIONS: Apart from morphological similarities, plastid genomes of holoparasitic plants also display other convergent features, such as rampant gene loss, biased nucleotide composition and accelerated evolutionary rates. In addition, the plastomes of Balanophoraceae have extremely low GC and gene content, and two unexpected changes in the genetic code. Limited data on the mitochondrial genomes of holoparasitic plants preclude thorough comparisons. Nonetheless, no obvious genomic features distinguish them from the mitochondria of free-living angiosperms, except for a higher incidence of HGT. HGT appears to be predominant in holoparasitic angiosperms with a long-lasting endophytic stage. Among the Balanophoraceae, mitochondrial genomes exhibit disparate evolutionary paths with notable levels of heteroplasmy in Rhopalocnemis and unprecedented levels of HGT in Lophophytum. Despite their differences, these Balanophoraceae share a multichromosomal mitogenome, a feature also found in a few free-living angiosperms.

Break-induced replication is the primary recombination pathway in plant somatic hybrid mitochondria: a model for mitochondrial horizontal gene transfer.

Somatic hybrids between distant species offer a remarkable model to study genomic recombination events after mitochondrial fusion. Recently, we described highly chimeric mitogenomes in two somatic hybrids between the Solanaceae Nicotiana tabacum and Hyoscyamus niger resulting from interparental homologous recombination. To better examine the recombination map in somatic hybrid mitochondria, we developed a more sensitive bioinformatic strategy to detect recombination activity based on high-throughput sequencing without assembling the hybrid mitogenome. We generated a new intergeneric somatic hybrid between N. tabacum and Physochlaina orientalis, and re-analyzed the somatic hybrids that we previously generated. We inferred 213 homologous recombination events across repeats of 2.1 kb on average. Most of them (~80%) were asymmetrical, consistent with the break-induced replication pathway. Only rare (2.74%) non-homologous events were detected. Interestingly, independent events frequently occurred in the same regions within and across somatic hybrids, suggesting the existence of recombination hotspots in plant mitogenomes. Break-induced replication is the main pathway of interparental recombination in somatic hybrid mitochondria. Findings of this study are relevant to mitogenome editing assays and to mechanistic aspects of DNA integration following mitochondrial DNA horizontal transfer events.

Nanoparticles Surface Chemistry Influence on Protein Corona Composition and Inflammatory Responses.

Nanoparticles are widely used for biomedical applications such as vaccine, drug delivery, diagnostics, and therapeutics. This study aims to reveal the influence of nanoparticle surface functionalization on protein corona formation from blood serum and plasma and the subsequent effects on the innate immune cellular responses. To achieve this goal, the surface chemistry of silica nanoparticles of 20 nm diameter was tailored via plasma polymerization with amine, carboxylic acid, oxazolines, and alkane functionalities. The results of this study show significant surface chemistry-induced differences in protein corona composition, which reflect in the subsequent inflammatory consequences. Nanoparticles rich with carboxylic acid surface functionalities increased the production of pro-inflammatory cytokines in response to higher level of complement proteins and decreased the number of lipoproteins found in their protein coronas. On another hand, amine rich coatings led to increased expressions of anti-inflammatory markers such as arginase. The findings demonstrate the potential to direct physiological responses to nanomaterials via tailoring their surface chemical composition.

Native and foreign mitochondrial and nuclear encoded proteins conform the OXPHOS complexes of a holoparasitic plant.

The intimate contact between the holoparasitic plant Lophophytum mirabile (Balanophoraceae) and its host plant (Fabaceae) facilitates the exchange of genetic information, increasing the frequency of horizontal gene transfer (HGT). Lophophytum stands out because it acquired a large number of mitochondrial genes (greater than 20) from its legume host that replaced the majority of the native homologs. These foreign genes code for proteins that form multisubunit enzyme complexes, such as those in the oxidative phosphorylation system (OXPHOS) and cytochrome c maturation (ccm) system, together with dozens of nuclear-encoded subunits. However, the existence and the origin of the nuclear subunits that form the major part of the OXPHOS and ccm system in Lophophytum remain unknown. It was proposed that nuclear-encoding genes whose products interact with foreign mitochondrial proteins are also foreign, minimizing the incompatibilities that could arise in the assembly and functioning of these multiprotein complexes. We identified a nearly complete set of OXPHOS and ccm system subunits evolving under selective constraints in the transcriptome of Lophophytum, indicating that OXPHOS is functional and resembles that of free-living angiosperms. Maximum Likelihood phylogenetic analyses revealed a single case of HGT in the nuclear genes, which results in mosaic OXPHOS and ccm system in Lophophytum. These observations raise new questions about the evolution and physiology of this parasitic plant. A putative case of cooperation between two foreign (one mitochondrial and one nuclear) genes is presented.

Ultra-small gold nanoclusters assembled on plasma polymer-modified zeolites: a multifunctional nanohybrid with anti-haemorrhagic and anti-inflammatory properties.

Hemostatic agents are pivotal for managing clinical and traumatic bleeding during emergency and domestic circumstances. Herein, a novel functional hybrid nanocomposite material consisting of plasma polymer-modified zeolite 13X and ultra-small gold nanoclusters (AuNCs) was fabricated as an efficient hemostatic agent. The surface of zeolite 13X was functionalised with amine groups which served as binding sites for carboxylate terminated AuNCs. Protein corona studies revealed the enhanced adsorption of two proteins, namely, coagulation factors and plasminogen as a result of AuNCs immobilization on the zeolite surface. The immune response studies showed that the hybrid nanocomposites are effective in reducing inflammation, which combined with a greater attachment of vitronectin, may promote wound healing. The hemostatic potential of the nanocomposite could be directly correlated with their immunomodulatory and anti-haemorrhagic properties. Together, the hybrid nanoengineered material developed in this work could provide a new avenue to tackle life-threatening injuries in civilian and other emergencies.

Transcriptional Landscape and Splicing Efficiency in Arabidopsis Mitochondria.

Plant mitochondrial transcription is initiated from multiple promoters without an apparent motif, which precludes their identification in other species based on sequence comparisons. Even though coding regions take up only a small fraction of plant mitochondrial genomes, deep RNAseq studies uncovered that these genomes are fully or nearly fully transcribed with significantly different RNA read depth across the genome. Transcriptomic analysis can be a powerful tool to understand the transcription process in diverse angiosperms, including the identification of potential promoters and co-transcribed genes or to study the efficiency of intron splicing. In this work, we analyzed the transcriptional landscape of the Arabidopsis mitochondrial genome (mtDNA) based on large-scale RNA sequencing data to evaluate the use of RNAseq to study those aspects of the transcription process. We found that about 98% of the Arabidopsis mtDNA is transcribed with highly different RNA read depth, which was elevated in known genes. The location of a sharp increase in RNA read depth upstream of genes matched the experimentally identified promoters. The continuously high RNA read depth across two adjacent genes agreed with the known co-transcribed units in Arabidopsis mitochondria. Most intron-containing genes showed a high splicing efficiency with no differences between cis and trans-spliced introns or between genes with distinct splicing mechanisms. Deep RNAseq analyses of diverse plant species will be valuable to recognize general and lineage-specific characteristics related to the mitochondrial transcription process.

Post-synthesis nanostructuration of BSA-Capsaicin nanoparticles generated by sucrose excipient.

In the pharmaceutical industry nano-hydrocolloid systems frequently coalesce or present nanoparticle aggregation after a long storage periods. Besides, the lyophilization process used to dry nanoparticles (NPs) produces loss of their original properties after dispersion. In this work we evaluated the effect on morphology and physicochemical properties of different protective excipients during drying of bovine serum albumin (BSA) NPs loaded with different concentrations of capsaicin. Capsaicin concentrations of 0, 812, 1625, 2437, and 3250 µg mL-1 were used; subsequently, NPs were dried with deionized water (DW), NaCl (DN), sucrose (DS), and not dried (ND). We found that ND, DW, and DN treatments showed a negative effect on the NPs properties; while, DS reduced the aggregation and produced the formation of isolated nanoparticles at higher concentrations of capsaicin (3250 µg mL-1), improving their circular shape, morphometrical parameters, and ζ-potential. The stability of the BSA-capsaicin NPs was associated to complex capsaicin/amino acid/water, in which GLY/GLN, ALA/HIS, ARG, THR, TYR, and Iso/CYS amino acids are involved in the restructuration of capsaicin molecules into the surface of nanoparticles during the drying process. The secondary nanostructuration in the post-synthesis stage can improve the molecular stability of the particles and the capacity of entrapping hydrophobic drugs, like capsaicin.

Horizontal gene transfers dominate the functional mitochondrial gene space of a holoparasitic plant.

Although horizontal gene transfer (HGT) is common in angiosperm mitochondrial DNAs (mtDNAs), few cases of functional foreign genes have been identified. The one outstanding candidate for large-scale functional HGT is the holoparasite Lophophytum mirabile, whose mtDNA has lost most native genes but contains intact foreign homologs acquired from legume host plants. To investigate the extent to which this situation results from functional replacement of native by foreign genes, functional mitochondrial gene transfer to the nucleus, and/or loss of mitochondrial biochemical function in the context of extreme parasitism, we examined the Lophophytum mitochondrial and nuclear transcriptomes by deep paired-end RNA sequencing. Most foreign mitochondrial genes in Lophophytum are highly transcribed, accurately spliced, and efficiently RNA edited. By contrast, we found no evidence for functional gene transfer to the nucleus or loss of mitochondrial functions in Lophophytum. Many functional replacements occurred via the physical replacement of native genes by foreign genes. Some of these events probably occurred as the final act of HGT itself. Lophophytum mtDNA has experienced an unprecedented level of functional replacement of native genes by foreign copies. This raises important questions concerning population-genetic and molecular regimes that underlie such a high level of foreign gene takeover.

Multichromosomal structure and foreign tracts in the Ombrophytum subterraneum (Balanophoraceae) mitochondrial genome.

Horizontal gene transfer (HGT) is frequent in parasitic plant mitochondria as a result of vascular connections established in host-parasite relationships. Recent studies of the holoparasitic plant Lophophytum mirabile (Balanophoraceae) revealed the unprecedented acquisition of a large amount of mitochondrial sequences from its legume host. We focused on a close relative, the generalist holoparasite Ombrophytum subterraneum, to examine the incidence of HGT events in the mitochondrial genome (mtDNA). The mtDNA of O. subterraneum assembles into 54 circular chromosomes, only 34 of which contain the 51 full-length coding regions. Numerous foreign tracts (totaling almost 100 kb, ~ 14% of the mtDNA), including 12 intact genes, were acquired by HGT from the Asteraceae hosts. Nine chromosomes concentrate most of those regions and eight are almost entirely foreign. Native homologs of each foreign gene coexist in the mtDNA and are potentially functional. A large proportion of shorter regions were related to the Fabaceae (a total of ~ 110 kb, 15.4%), some of which were shared with L. mirabile. We also found evidence of foreign sequences donated by angiosperm lineages not reported as hosts (Apocynaceae, Euphorbiaceae, Lamiaceae, and Malvales). We propose an evolutionary hypothesis that involves ancient transfers from legume hosts in the common ancestor of Ombrophytum and Lophophytum followed by more recent transfer events in L. mirabile. Besides, the O. subterraneum mtDNA was also subjected to additional HGT events from diverse angiosperm lineages, including large and recent transfers from the Asteraceae, and also from Lamiaceae.

The Influence of Nanoparticle Shape on Protein Corona Formation.

Nanoparticles have become an important utility in many areas of medical treatment such as targeted drug and treatment delivery as well as imaging and diagnostics. These advances require a complete understanding of nanoparticles' fate once placed in the body. Upon exposure to blood, proteins adsorb onto the nanoparticles surface and form a protein corona, which determines the particles' biological fate. This study reports on the protein corona formation from blood serum and plasma on spherical and rod-shaped nanoparticles. These two types of mesoporous silica nanoparticles have identical chemistry, porosity, surface potential, and size in the y-dimension, one being a sphere and the other a rod shape. The results show a significantly larger amount of protein attaching from both plasma and serum on the rod-like particles compared to the spheres. Interrogation of the protein corona by liquid chromatography-mass spectrometry reveals shape-dependent differences in the adsorption of immunoglobulins and albumin proteins from both plasma and serum. This study points to the need for taking nanoparticle shape into consideration because it can have a significant impact on the fate and therapeutic potential of nanoparticles when placed in the body.

The complete organelle genomes of Physochlaina orientalis: Insights into short sequence repeats across seed plant mitochondrial genomes.

Short repeats (SR) play an important role in shaping seed plant mitochondrial genomes (mtDNAs). However, their origin, distribution, and relationships across the different plant lineages remain unresolved. We focus on the angiosperm family Solanaceae that shows great variation in repeat content and extend the study to a wide diversity of seed plants. We determined the complete nucleotide sequences of the organellar genomes of the medicinal plant Physochlaina orientalis (Solanaceae), member of the tribe Hyoscyameae. To understand the evolution of the P. orientalis mtDNA we made comparisons with those of five other Solanaceae. P. orientalis mtDNA presents the largest mitogenome (∼685 kb in size) among the Solanaceae and has an unprecedented 8-copy repeat family of ∼8.2 kb in length and a great number of SR arranged in tandem-like structures. We found that the SR in the Solanaceae share a common origin, but these only expanded in members of the tribe Hyoscyameae. We discuss a mechanism that could explain SR formation and expansion in P. orientalis and Hyoscyamus niger. Finally, the great increase in plant mitochondrial data allowed us to systematically extend our repeat analysis to a total of 136 seed plants to characterize and analyze for the first time families of SR among seed plant mtDNAs.

Elucidating genomic patterns and recombination events in plant cybrid mitochondria.

KEY MESSAGE: Cybrid plant mitochondria undergo homologous recombination, mainly BIR, keep a single allele for each gene, and maintain exclusive sequences of each parent and a single copy of the homologous regions. The maintenance of a dynamic equilibrium between the mitochondrial and nuclear genomes requires continuous communication and a high level of compatibility between them, so that alterations in one genetic compartment need adjustments in the other. The co-evolution of nuclear and mitochondrial genomes has been poorly studied, even though the consequences and effects of this interaction are highly relevant for human health, as well as for crop improvement programs and for genetic engineering. The mitochondria of plants represent an excellent system to understand the mechanisms of genomic rearrangements, chimeric gene formation, incompatibility between nucleus and cytoplasm, and horizontal gene transfer. We carried out detailed analyses of the mtDNA of a repeated cybrid between the solanaceae Nicotiana tabacum and Hyoscyamus niger. The mtDNA of the cybrid was intermediate between the size of the parental mtDNAs and the sum of them. Noticeably, most of the homologous sequences inherited from both parents were lost. In contrast, the majority of the sequences exclusive of a single parent were maintained. The mitochondrial gene content included a majority of N. tabacum derived genes, but also chimeric, two-parent derived, and H. niger-derived genes in a tobacco nuclear background. Any of these alterations in the gene content could be the cause of CMS in the cybrid. The parental mtDNAs interacted through 28 homologous recombination events and a single case of illegitimate recombination. Three main homologous recombination mechanisms were recognized in the cybrid mitochondria. Break induced replication (BIR) pathway was the most frequent. We propose that BIR could be one of the mechanisms responsible for the loss of the majority of the repeated regions derived from H. niger.

Self-sterilizing antibacterial silver-loaded microneedles.

Microneedle patches have become an exciting means for transdermal delivery of various therapeutics. Herein, we report on self-sterilizing dissolving nanosilver-loaded microneedle patches created from carboxymethylcellulose capable of suppressing microbial pathogen growth at the insertion site.

Starch accumulation is associated with active growth in A. tequilana.

Transcriptome analysis of different tissues and developmental stages of A. tequilana plants led to the identification of full length cDNAs and the corresponding amino acid sequences for enzymes involved in starch metabolism in this species. Comparison with sequences from other species confirmed the identities of putative A. tequilana starch metabolism genes and uncovered differences in the evolutionary patterns of these genes between gramineous and non-gramineous monocotyledons. In silico expression patterns showed high levels of expression of starch metabolism genes in shoot apical meristem tissue and histological studies showed the presence of starch in leaf primordia surrounding the shoot apical meristem and in the primary thickening meristem of the stem. Starch was also found to accumulate significantly in developing floral organs and immature embryos. Low levels of starch were observed overall in leaf tissue with the exception of stomatal guard cells where starch was abundant. In root tissue, starch was only observed in statoliths at the root tip. A. tequilana starch grains were found to be small in comparison to other species and have an almost spherical form. The data for gene expression and histological localization are consistent with a role for starch as a transient carbohydrate store for actively growing tissues in A. tequilana.

Protein Interactions with Nanoengineered Polyoxazoline Surfaces Generated via Plasma Deposition.

Protein adsorption to biomaterials is critical in determining their suitability for specific applications, such as implants or biosensors. Here, we show that surface nanoroughness can be tailored to control the covalent binding of proteins to plasma-deposited polyoxazoline (PPOx). Nanoengineered surfaces were created by immobilizing gold nanoparticles varying in size and surface density on PPOx films. To keep the surface chemistry consistent while preserving the nanotopography, all substrates were overcoated with a nanothin PPOx film. Bovine serum albumin was chosen to study protein interactions with the nanoengineered surfaces. The results demonstrate that the amount of protein bound to the surface is not directly correlated with the increase in surface area. Instead, it is determined by nanotopography-induced geometric effects and surface wettability. A densely packed array of 16 and 38 nm nanoparticles hinders protein adsorption compared to smooth PPOx substrates, while it increases for 68 nm nanoparticles. These adaptable surfaces could be used for designing biomaterials where proteins adsorption is or is not desirable.

Unparalleled replacement of native mitochondrial genes by foreign homologs in a holoparasitic plant.

Horizontal gene transfer (HGT) among flowering plant mitochondria occurs frequently and, in most cases, leads to nonfunctional transgenes in the recipient genome. Parasitic plants are particularly prone to this phenomenon, but their mitochondrial genomes (mtDNA) have been largely unexplored. We undertook a large-scale mitochondrial genomic study of the holoparasitic plant Lophophytum mirabile (Balanophoraceae). Comprehensive phylogenetic analyses were performed to address the frequency, origin, and impact of HGT. The sequencing of the complete mtDNA of L. mirabile revealed the unprecedented acquisition of host-derived mitochondrial genes, representing 80% of the protein-coding gene content. All but two of these foreign genes replaced the native homologs and are probably functional in energy metabolism. The genome consists of 54 circular-mapping chromosomes, 25 of which carry no intact genes. The likely functional replacement of up to 26 genes in L. mirabile represents a stunning example of the potential effect of rampant HGT on plant mitochondria. The use of host-derived genes may have a positive effect on the host-parasite relationship, but could also be the result of nonadaptive forces.

Psychometric Properties of the Mexican Version of the Mindful Attention Awareness Scale (MAAS) / Propiedades psicométricas de la versión mexicana de la escala de atención Plena (MAAS)

This study presents a translation of the Mindful Attention Awareness Scale (MAAS) into Mexican Spanish, and examines its psychometric properties as well as its relationship with socio-demographic variables. The MAAS measures the frequency with which people experience mindful states. A sample of N = 622 healthy adult Mexicans completed the MAAS. A smaller sub-sample (n=195) completed the Five-Facet Mindfulness Questionnaire (FFMQ), a well-known mindfulness measure, to obtain concurrent validity. Exploratory factor analysis (EFA) revealed a one-factor solution, and reliability coefficients were adequate. Confirmatory factor analysis (CFA) showed adequate goodness of fit indexes. Moreover, relationships between Mexican Spanish MAAS scores and socio-demographic variables were also explored, and differences between-groups were found in mean scores both in alcohol consumers and religious practitioners. No other significant differences between groups were found. Results suggest that the Mexican version of the MAAS is a reliable and valid instrument to use with a healthy adult Mexican sample.

Este estudio presenta una traducción de la Mindful Attention Awareness Scale (Escala de Atención Plena) al español mexicano y examina sus propiedades psicométricas, así como su relación con variables sociodemográficas. La Escala de Atención Plena mide la frecuencia con la que las personas experimentan estados de atención plena. Una muestra de N=622 adultos mexicanos sanos completaron la Escala de Atención Plena. Una submuestra más pequeña (n=195) completó el Cuestionario de Cinco Facetas de la Atención Plena, el cual es una escala bien conocida de atención plena, para obtener validez concurrente. El análisis factorial exploratorio reveló una solución unifactorial y los coeficientes de confiabilidad fueron adecuados. El análisis factorial confirmatorio mostró índices adecuados de bondad de ajuste. Además, también se exploraron las relaciones entre la versión Mexicana del MAAS y variables sociodemográficas, encontrándose diferencias entre grupos tanto en consumidores de alcohol como en practicantes de religión. No se encontraron otras diferencias significativas entre grupos. Los resultados sugieren que la versión mexicana de la MAAS es un instrumento confiable y válido para utilizarse con población mexicana adulta y sana.

Gastric tissue damage analysis generated by ischemia: bioimpedance, confocal endomicroscopy, and light microscopy.

The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

Cirugía endoscópica de oído: aplicaciones / Endoscopic surgery of the ear: applications

La tecnología de endoscopia, de uso común en cirugía de senos paranasales, ha ganado espacio en otología, en especial para procedimientos de oído crónico mediante abordaje transcanal mínimamente invasivo. En la actualidad, los endoscopios de cuerpo rígido permiten nuevas aplicaciones en la visualización de la anatomía y patología del oído medio, que no es fácil obtener con microscopio. Del mismo modo, su aplicación se puede extender a cirugías neurotológicas. En el presente estudio realizamos una breve descripción de la utilización más común de los endoscopios en cirugía otológica...

The endoscopy technology, commonly used in nasal sinus surgery, has gained acceptance in otology specially for chronic ear procedures through transcanal approach minimally invasive. Rigid endoscopes allow new applications in middle ear anatomy and pathology view that is no easy to obtain with microscope. In the same way, its application can be extended to neurotological surgeries. In the present document, we make a brief description of the most common uses of endoscopes in otological surgery...

Olfactory conditioning in the third instar larvae of Drosophila melanogaster using heat shock reinforcement.

Adult Drosophila melanogaster has long been a popular model for learning and memory studies. Now the larval stage of the fruit fly is also being used in an increasing number of classical conditioning studies. In this study, we employed heat shock as a novel negative reinforcement for larvae and obtained high learning scores following just one training trial. We demonstrated heat-shock conditioning in both reciprocal and non-reciprocal paradigms and observed that the time window of association for the odor and heat shock reinforcement is on the order of a few minutes. This is slightly wider than the time window for electroshock conditioning reported in previous studies, possibly due to lingering effects of the high temperature. To test the utility of this simplified assay for the identification of new mutations that disrupt learning, we examined flies carrying mutations in the dnc gene. While the sensitivity to heat shock, as tested by writhing, was similar for wild type and dnc homozygotes, dnc mutations strongly diminished learning. We confirmed that the learning defect in dnc flies was indeed due to mutation in the dnc gene using non-complementation analysis. Given that heat shock has not been employed as a reinforcement for larvae in the past, we explored learning as a function of heat shock intensity and found that optimal learning occurred around 41 °C, with higher and lower temperatures both resulting in lower learning scores. In summary, we have developed a very simple, robust paradigm of learning in fruit fly larvae using heat shock reinforcement.