Developing robust behavioral assays to study olfactory-driven behaviors allows for greater insight into the neural mechanisms behind them. Oftentimes, olfactory behavioral assays require a two-choice design, consistent variables, and controlled stimulus application. This can be challenging when working with marine dwelling organisms such as nudibranchs. Extensive work shows the importance of olfaction in both pre-metamorphic larval development and adult stage nudibranchs (Gastropoda, Mollusca). However, there is little research investigating how olfaction plays a role in rapidly developing, post-metamorphic juvenile nudibranchs such as Berghia stephanieae. To study olfactory-associated behaviors in juvenile Berghia, a novel behavioral microfluidic chip was designed which met the requirements for a reliable olfaction assay. Baseline motor behaviors such as general locomotion, turns, and contractions were observed. Despite individuals being raised in the same cohort, animal-to-animal variability was found in relation to their baseline behaviors. Xylene cyanol, a commonly used dye in microfluidics, was found to be aversive at a range of concentrations (0.075% - 0.025%) causing the animal to tightly contract and turn away from the stimulus. Juveniles showed no significant behavioral response to a 0.00125% dilution which was necessary to confirm a constant flow in the microfluidic chip. Juveniles showed a strong preference towards fluid that had been conditioned with their preferred food source odor, the sea anemone Exaiptasia diaphana. This was judged to be an innate preference because it occurred upon the first exposure of these animals to the odor. Serial dilution of Exaiptasia conditioned seawater (ECS) uncovered a threshold for behavioral preference of 60%. In summary, the development of this behavioral provides an opportunity to present controlled olfactory stimuli while observing the juvenile’s behaviors. This will allow future experiments to examine the neural mechanisms behind both aversive and attractive stimuli and provides a method for testing olfactory learning and memory in this species.

Across the animal kingdom, predation is a ubiquitous and omnipresent selective agent for a variety of traits. I aimed to address gaps in our knowledge pertaining to how predation shapes animal behavior. Many species of fish naturally occur in drainages that differ in the density of predators and exhibit obvious population divergence, making them ideal study organisms to investigate predator-driven behavioral evolution. In Chapter 1, I conducted a systematic review of the literature. The purpose of this review was to determine if predation acted as a stronger or weaker selective agent on particular behavioral traits (e.g., foraging, mating, antipredator etc.) across fish. This review showed that predation does not always drive behavior in predictable ways, and that some behavioral traits more consistently diverge than others. It was evident that antipredator behaviors are extremely variable but were typically measured in response to a visual stimulus. Investigations on intraspecific variation pertaining to how fish acquire, process, and respond to information across other sensory modalities are needed. To address this, I focused on a Neotropical fish, Brachyrhaphis rhabdophora (Poeciliidae), from Costa Rica that occur in distinct predation environments. For Chapter 2, I evaluated whether males and females exhibit differential responses to conspecific chemical alarm cues. Chemical alarm cues are released when a prey is injured by a predator and are an honest indicator of risk. It was clear that B. rhabdophora responded to alarm cues, but that males and females sometimes employed different antipredator strategies depending on what predation environment they were from. However, we know that in group-living species, such as B. rhabdophora, risk information can also be acquired indirectly through social cues. There are tradeoffs associated with relying on direct vs. indirect information, and these sources of information may sometimes conflict. For Chapter 3 I considered how B. rhabdophora integrates conflicting information to elicit antipredator behavior. I again exposed fish directly to chemical alarm cues and measured how their antipredator responses changed when visually observing conflicting or reinforcing social information. I found that individuals integrated personal and social information differently based on their evolutionary history with predators. Further, we found evidence that even a single observer fish is able to influence group behavior. Finally, for Chapter 4, I evaluated sex-specific variation in brain size across predation environments. According to the "expensive-tissue hypothesis" there should only be investment in brain tissue when there is sufficient selection for enhanced cognitive abilities. Prey under elevated selection from predators should invest more in cognitive traits to enhance survival, but how sex interplays with this effect is unclear. I found that females had higher relative total brain volumes than males, but males exhibited more variation across predation environments in the relative volumes for certain brain regions. This work as a whole suggests that, yes, evolutionary history matters for a variety of sensory-related traits in B. rhabdophora.

Le système trigéminal –tout comme l’olfaction et la gustation– est un sens chimique qui permet la perception des informations chimiosensorielles de notre environnement. Contrairement à l’olfaction et à la gustation, notre connaissance du traitement des mélanges par le système trigéminal est limitée. Nous avons donc utilisé des mélanges de trois agonistes relativement spécifiques à des récepteurs (eucalyptol, agoniste TRPM8; aldéhyde cinnamique, agoniste TRPA1 ; camphre, agoniste TRPV1) et d’une odeur pure (alcool phényléthylique) dans différentes proportions afin de déterminer les dimensions de base de la perception trigéminale. Quatre dimensions principales se sont avérées pertinentes: l’intensité, la sensation de chaleur, la sensation de froid et la douleur. Nous avons utilisé ces dimensions pour étudier la perception de mélanges et de combinaisons dans différentes proportions d’un stimulus qui procure une sensation de froid (eucalyptol) et d’un stimulus qui procure une sensation de chaleur (aldéhyde cinnamique). Les résultats indiquent que les mélanges obtiennent généralement des scores plus élevés que les combinaisons sur les dimensions « intensité », « sensation de chaleur » et « douleur » alors que les combinaisons obtiennent des scores plus élevés sur la dimension « sensation de froid ». Ces résultats suggèrent des interactions spécifiques pour les différentes dimensions de la perception trigéminale. Nous en venons à la conclusion d’un effet d’additivité pour les mélanges sur les dimensions « intensité », « sensation de chaleur » et « douleur » alors que nous observons plutôt un effet de suppression de la perception de froid pour les deux stimuli dans les mélanges, ce qui semble indiquer des interactions particulières pouvant prendre place aux niveaux périphérique ou central. / The trigeminal system – along with smell and taste – is a chemical sense that allows the perception of chemosensory information in our environment. However, contrary to smell and taste, we lack knowledge of the trigeminal processing of mixtures. We therefore investigated trigeminal perception using mixtures of three relatively receptor specific agonists (eucalyptol, TRPM8 agonist; cinnamaldehyde, TRPA1 agonist and camphor, TRPV1 agonist) together with one control odor (phenyl ethyl alcohol or PEA) in different proportions to determine basic dimensions of trigeminal perception. Four main dimensions were proven relevant to trigeminal perception: intensity, warmth sensation, cold sensation and pain sensation. Subsequently, we used these dimensions to investigate perception of mixtures and combinations of trigeminal stimuli using different concentrations of a cooling stimulus (eucalyptol) with a stimulus which evokes warmth perception (cinnamaldehyde). Results showed mixtures generally yielded higher ratings than combinations on the trigeminal dimensions “intensity”, “warm” and “painful” whereas combinations yielded higher ratings than mixtures on the trigeminal dimension “cold”. These results suggest dimension specific interactions in the perception of trigeminal mixtures. For the mixtures we used, we reach the conclusion of additivity for the dimensions “intensity”, “warm” and “painful”; we observed suppression of the cold perception of both stimuli in mixtures suggesting particular interactions which may take place on peripheral or central levels.

The present study aimed to investigate intranasal trigeminal sensitivity in a large sample of patients with anosmia due to different etiologies. We investigated the trigeminal detection threshold for formic acid in healthy controls (n = 96) and patients with anosmia due to head trauma (n = 18) or sinonasal disease (n = 54). Anosmics exhibited higher thresholds compared with normosmics (p < 0.001). In addition, thresholds were found to be higher in patients with posttraumatic anosmia compared to anosmics with sinonasal disease (p < 0.001). The data indicate that (1) loss of olfactory sensitivity in humans may be associated with a decreased sensitivity towards trigeminal stimuli and (2) alteration of intranasal trigeminal function is stronger in patients with posttraumatic anosmia compared to patients with sinonasal disease. This may have implications for the medicolegal investigation of anosmic patients where trigeminal stimuli are frequently used to assess the patient’s response bias. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Les capacités cognitives pour assurer l’exploration et la découverte de nouvelles niches écologiques sont au cœur des processus d’adaptation et de survie des espèces vertébrés et invertébrés. A cet égard, les systèmes neuronaux chimio-sensoriels composés des organes olfactifs et gustatifs permettent le guidage et repérage des sources de nourritures et/ou des partenaires sexuels. Un fait marquant chez les insectes et en particulier la drosophile réside dans le fait que les organes gustatifs sont disséminés sur le corps. La bordure antérieure de l’aile est tapissée avec des sensilles gustatives alternées avec des sensilles mécaniques. La fonctionnalité et le rôle des cellules gustatives au niveau de l’aile de la drosophile reste énigmatique et à ce jour largement inconnue (Stocker, 1994). Notre travail a consisté à explorer la signalisation et le mécanisme de transduction de ces récepteurs et à questionner leur importance dans l’adaptation des insectes à leur écosystème. Nos résultats portent sur trois volets. Nous avons vérifié que l’expression des récepteurs du goût est effective dans les ailes des trois insectes différents (drosophiles, pucerons et abeilles) par RT-PCR. Nous avons ensuite étudié la fonctionnalité de ces récepteurs vis-à-vis des molécules sucrées et amères à l’aide d’une souche transgénique (G-CaMP), qui exhibe une forte fluorescence provoquée par des piques de calcium cytosolique. Enfin, des tests comportementaux ont été réalisé avec une souche transgénique (Poxn*) dans laquelle les sensilles chimio-sensorielles de l’aile sont spécifiquement invalidés sans altérer les autres structures olfactives et/ou gustatives. Les résultats montrent un effet significatif des cellules chimio-sensorielles de l’aile quant à l’orientation dans l’espace et à l’apprentissage Bayesien. Nos résultats sur ces trois volets nous ont permis d’élaborer des hypothèses au regard de l’évolution neuroanatomique de l’aile des insectes depuis les organismes ancestraux d’origine marine desquels ils dérivent. Des experts en aérodynamiques proposent la création d’un vortex durant le vol qui forme une spirale de courant d’air le long de la bordure antérieur de l’aile. La parfaite superposition entre ce vortex et le nerf costal de l’aile nous permet de déduire que les vibrations de l’aile entre 50 et 1.000 Hertz chez les insectes sont en mesure de nébuliser des matériaux (micro poussières, micro gouttelettes, molécules faiblement volatiles) lesquels vont être captés/entrainés dans le vortex et adressés aux sensilles gustatives. Notre hypothèse est que ce mécanisme permettrait aux insectes pollinisateurs de gouter les fleurs sans se poser et sans mettre à contribution la trompe buccale (proboscis). Ce scénario permettrait de dissocier le goût de l’ingestion digestive en évitant les empoisonnements par des molécules toxiques émises par les plantes et d’autre part il rend l’exploration plus efficace, en minimisant le temps de recherche. / Cognitive capacities used to ensure the exploration and discovery of new ecological niches are at the heart of the process of adaptation and survival of vertebrate and invertebrate species. In this respect, the neural chemosensory systems, composed of the olfactory and gustatory organs, allow the guidance and finding of food sources and/or sexual partners. A striking feature in insects and particularly in Drosophila is that gustatory organs are disseminated in the body. The anterior margin of the wing is lined with gustatory sensilla alternated with mechanosensory sensilla. The function of gustatory cells in the wing of Drosophila remains enigmatic and actually quite unknown (Stocker, 1994). Our work consisted in exploring the signaling and the transduction mechanisms of these receptors and in questioning their importance in the adaptation of insects to their ecosystem. Our results are based on three components. We have verified that the expression of gustatory receptors occurs in the wings of three different insects (Drosophila, aphid and honey bee) by RT-PCR. We have studied the function of these receptors vis-à-vis of sweet and bitter molecules using a transgenic line (G-CaMP) that exhibits a strong fluorescence provoked by cytosolic calcium picks. Finally, behavioral assays have been realized with a transgenic line (Poxn*) in which the chemosensory sensilla have been invalidated without altering the other olfactory and gustatory structures. Our results show a significant effect of wing chemosensory cells as far as orientation is space and Bayesian learning and have permitted us to elaborate hypothesis regarding the neuroanatomical evolution of the wing of insects since ancestral organisms of marine origin from which they derive. Experts in aerodynamics propose the creation of a vortex during flight that forms a spiral of air along the anterior border of the wing. The perfect superposition between this vortex and the costal nerve of the wing allows us to deduce that the vibrations of the insect wing between 50 and 1.000 Hertz are able to nebulize materials (microdust, microdrops, weakly volatile molecules), which are captured/trapped in the vortex and addressed to the gustatory sensilla. Our hypothesis is that this mechanism would let pollinator insects taste flowers without landing and without involving the proboscis. In this scenario insects would dissociate taste from ingestion, avoiding poisoning by toxic molecules emitted by plants and rending exploration more efficient by minimizing searching time.

Le corps humain comporte cinq sens dont les rôles principaux sont d’assurer la santé et la sécurité de l’individu. Parmi ceux-ci, le goût et l’odorat font partie du système chimiosensoriel, c’est-à-dire que ce sont deux sens détectent les particules chimiques afin d’envoyer l’information au système nerveux central. Il est connu qu’une diminution de l’efficacité de l’odorat engendrée par différentes situations comme l’obésité ou le vieillissement peut avoir des répercussions négatives importantes sur la santé. L’activité physique représente une intervention pour laquelle les bienfaits sont déjà bien connus pour prévenir entre autres des maladies chroniques, mais ses mécanismes reliés à l’odorat et aux odeurs restent toujours à être explorés. Ce mémoire a donc pour objectif d’approfondir la compréhension des impacts de l’activité physique sur l’odorat et des impacts de la stimulation des odeurs sur des composantes clés de la pratique de l’activité physique via la réalisation de deux revues systématiques. Premièrement, l’activité physique aiguë et chronique n’ont pas la même incidence sur la détection des odeurs. En effet, l’activité physique chronique améliore clairement la réponse olfactive, alors que l’activité physique aiguë démontre des résultats moins concluants. Deuxièmement, la stimulation olfactive génère des résultats qui diffèrent d’une odeur à l’autre et d’un type d’activité à l’autre (c.-à-d., force, cardiovasculaire, précision, équilibre). Parmi les odeurs les plus testées, la menthe et la lavande améliorent de façon générale les sensations perçues lors de la pratique d’activité physique, modulant ainsi quelques mécanismes physiologiques comme la pression artérielle. À la lumière de ces résultats, une pratique régulière d’activité physique est favorable à la protection de l’odorat dans le but de limiter les risques reliés à une perte olfactive. De plus, la stimulation olfactive basée sur certaines odeurs spécifiques comme la menthe ou la lavande peut être une solution envisageable pour bonifier la réponse à l’effort. / The human body possesses five senses which have the primary role of ensuring the health and safety of the individual. Among these senses, taste and smell are part of the chemosensory system, meaning that they can detect chemical particles to transmit information to the central nervous system. It is known that a decrease in the efficiency of the sense of smell due to various factors such as obesity or aging can have significant negative impacts on health. Physical activity represents an intervention for which the benefits are already well known in preventing chronic diseases, but its mechanisms related to smell and odors remain to be explored. This thesis therefore aims to deepen the understanding of the impacts of physical activity on smell and the impacts of odor stimulation on key components of physical activity practice through the realization of two systematic reviews. Firstly, acute and chronic physical activity do not have the same impact on odor detection. Indeed, chronic physical activity clearly improves this olfactory variable, while acute physical activity shows less conclusive results. Secondly, olfactory stimulation generates results that differ from one odor to another and from one type of activity to another (i.e., strength, cardiovascular, precision, balance). Among the most tested odors, mint and lavender generally improve the sensations perceived during the practice of physical activity, thus modulating some physiological mechanisms such as blood pressure. Based on these findings, regular physical activity is favorable to protecting the sense of smell in order to limit the risks related with olfactory loss. In addition, olfactory stimulation based on certain specific odors such as mint or lavender can be a viable solution to improve the response to effort.

The Myxococcus xanthus Dif and Frz chemosensory pathways play important roles in the regulation of gliding motility. The Dif system regulates the production of exopolysaccheride (EPS), which is essential for social motility and fruiting body formation. The Frz pathway controls reversal frequency, which is fundamental for directed movement by this surface-gliding bacterium. In addition, both pathways are involved in the chemotactic response towards several phosphatidylethanolamine (PE) species such that the Dif pathway is required for excitation while the Frz pathway is essential for adaptation. In this study we addressed three crucial questions regarding the signal processing of these two chemosensory pathways by focusing on DifA and FrzCD, the MCP homologs from their respective pathways. First, the receptor protein in the Dif pathway, DifA, lacks a perisplasmic domain, the typical signal-sensing structure. To examine whether DifA shares similar transmembrane signaling mechanism with typical transmembrane sensor proteins (MCPs and sensor kinases), we constructed a chimeric protein that is composed of the N-terminus of NarX (nitrate sensor kinase) and the C-terminus of DifA. This NarX-DifA chimera restores the DifA functionality (EPS production, agglutination, S-motility and development) to a "difA mutant in a nitrate-dependent manner, suggesting DifA shares a similar transmembrane signaling mechanism with typical MCPs and sensor kinases despite its unorthodox structure. Second, the M. xanthus chemotaxis is still controversial. It has been argued that the taxis-like response in this slowly gliding bacterium could result from physiological effects of certain chemicals. To study motility regulation by the Frz pathway, we constructed two chimeras between the N-terminus of NarX and C-terminus of FrzCD, which is the receptor protein of the Frz pathway. The two chimeras, NazDF and NazDR, are identical except that NazDR contains a G51R mutation in the otherwise wild-type NarX sensory module. This G51R mutation was shown to reverse the signaling output of a NarX-Tar chimera to nitrate. We discovered that nitrate specifically decreased the reversal frequency of NazDF-expressing cells and increased that of NazDR-expressing cells. These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazDR strain failed to adapt to nitrate in temporal assays, as did the wild type to known repellents. Therefore, the lack of temporal adaptation to negative stimuli is an intrinsic property in M. xanthus motility regulation. Third, the Dif and Frz pathways are both involved in the chemotactic response towards certain PE molecules such that the Dif pathway is required for excitation and while the Frz system is essential for adaptation. In addition, 12:0 PE, known to be sensed by DifA, results in increased FrzCD methylation. These findings suggested that in the regulation of PE response, two pathways communicate with each other to mediate adaptation. Here we provided evidence to indicate that DifA does not undergo methylation during EPS regulation and PE chemotaxis. On the other hand, using mutants expressing the NarX-DifA chimera, it was found that signal transduction through DifA, DifC (CheW-like) and DifE (CheA-like) modulates FrzCD methylation. Surprisingly, the attractant 12:0 PE can modulate FrzCD methylation in two ways distinguishable by the dependency on DifA, DifC and DifE. The DifACE-independent mechanism, which may result from specific sensing of 12:0 PE by FrzCD, increases FrzCD methylation as expected. Unexpectedly, 12:0 PE decreases FrzCD methylation with the DifACE-dependent mechanism. This "opposite" FrzCD methylation by DifACE-dependent signaling was supported by results from NafA-expressing mutants because nitrate, which acts as a repellent, increases FrzCD methylation. Based on these findings, we proposed a model for chemotaxis toward 12:0 PE (and 16:1 PE). In this model, DifA and FrzCD both sense the same signal and activate the pathways of excitation (Dif) and adaptation (Frz) independently. The two pathways communicate with each other via methylation crosstalk between DifACE and FrzCD in such a way that processes of excitation and adaptation can be coordinated. / Ph. D.

The freshwater mussel fauna of the North Fork Holston River (NFHR) downstream of Saltville, VA declined from at least 24 species, as observed in 1918, to one species in 1974 due to mercury pollution. To determine the degree of recovery of mussels in the NFHR downstream of Saltville, and to provide recommendations for future mussel translocation sites, 19 sites were surveyed using a snorkeling catch-per-unit-effort (CPUE) method. At sites where investigator CPUE values (no./h) equaled or exceeded 5 mussels/h, a CPUE survey was conducted along transect lines. If investigator CPUE values equaled 10 mussels/h, a quadrat survey was conducted along the transects. Nine species of mussels were observed in the NFHR, and reproduction, as indicated by the presence of juveniles, was noted at 5 sites. Recovery of mussels was judged to be occurring downstream of NFHRM 56.4 based on species aggregations and recruitment. The number of mussels collected at sites, random CPUE (no./h), transect CPUE (no./h), and density (no./O.25m2) were generally inversely correlated to total Hg content, but not methyl mercury content, as measured in Corbicula fluminea from proximate sites. Random and transect CPUE (no./h) were found to be poor predictors of site densities (no./O.25m2) and popUlation estimates. Translocation recommendations for the NFHR downstream of Saltville, VA were made based on species aggregations, recruitment, and the distribution of total mercury. / Master of Science

The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW₃ proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.

The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of G-protein-coupled olfactory receptors (ORs) and olfactory carriers. Two such carrier gene families are found in insects: the odorant binding proteins (OBPs) and the chemosensory proteins (CSPs). In olfactory sensilla, OBPs and CSPs are believed to deliver hydrophobic air-borne molecules to ORs, but their expression in non-olfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes. ¶ Bioinformatics and experimental approaches were used to characterise the OBP and CSP gene families in a highly social insect, the western honey bee (Apis mellifera). Comparison with other insects reveals that the honey bee has the smallest set of these genes, consisting of only 21 OBPs and 6 CSPs. These numbers stand in stark contrast to the 66 OBPs and 7 CSPs in the mosquito Anopheles gambiae and the 46 OBPs and 20 CSPs in the beetle Tribolium castaneum. The genes belonging to both families are often organised in clusters, and evolve by lineage specic expansions. Positive selection has been found to play a role in generating a greater sequence diversication in the OBP family in contrast to the CSP gene family that is more conserved, especially in the binding pocket. Expression proling under a wide range of conditions shows that, in the honey, bee only a minority of these genes are antenna-specic. The remaining genes are expressed either ubiquitously, or are tightly regulated in specialized tissues or during development. These findings support the view that OBPs and CSPs are not restricted to olfaction, and are likely to be involved in broader physiological functions. ¶ Finally, the detailed expression study and the functional characterization of a member of the CSP family, uth (unable-to-hatch), is reported. This gene is expressed in a maternal-zygotic fashion, and is restricted to the egg and embryo. Blocking the zygotic expression of uth with double-stranded RNA causes abnormalities in all body parts where this gene is highly expressed. The treated embryos are `unable-to-hatch' and cannot progress to the larval stages. Our ndings reveal a novel, essential role for this gene family and suggest that uth is an ectodermal gene involved in embryonic cuticle formation.