Gene Expression In Larvae Research Articles

Beyond adult models: Tribolium castaneum larval timekeeping reveals unexpected robustness and insights into circadian clock.

Circadian rhythms are self-sustained endogenous oscillations that are found in all living organisms. In insects, circadian rhythms control a wide variety of behavioral and physiological processes, including feeding, locomotion, mating, and metabolism. While the role of circadian rhythms in adult insects is well-understood, it is largely unexplored in larvae. This study investigates the potential for larval synchronized activity in the red flour beetle (Tribolium castaneum), a species exhibiting solitary and aggregation phases. We hypothesized that, similar to adults, larvae would exhibit a daily activity pattern governed by an endogenous circadian clock. We further predicted that the transition between the solitary and gregarious phases extends to unique temporal activity patterns. Our results revealed unique timekeeper gene expression in larvae, leading to a distinct daily rhythm characterized by nocturnal activity. Cues indicating on potential cannibalism did not change daily activity peak. However, the absence of these cues significantly reduced the proportion of rhythmic larvae and led to higher variation in peak activity, highlighting the crucial role of social interactions in shaping their rhythmicity. This study sheds light on the evolution and function of larval synchronization in group-living insects, offering novel insights into this complex behavior.

Immunomodulatory effects of a probiotic combination treatment to improve the survival of Pacific oyster (Crassostrea gigas) larvae against infection by Vibrio coralliilyticus.

The culture of Pacific oysters (Crassostrea gigas) is of significant socio-economic importance in the U.S. Pacific Northwest and other temperate regions worldwide, with disease outbreaks acting as significant bottlenecks to the successful production of healthy seed larvae. Therefore, the current study aims to describe the mechanisms of a probiotic combination in improving the survival of C. gigas larvae. Specifically, we investigate changes in C. gigas larval gene expression in response to V. coralliilyticus infection with or without a pre-treatment of a novel probiotic combination. Treatment groups consisted of replicates of Pacific oyster larvae exposed to a) a combination of four probiotic bacteria at a total concentration of 3.0 x 105 CFU/mL at 18 hours post-fertilization (hpf), b) pathogenic V. coralliilyticus RE22 at a concentration of 6.0 x 103 CFU/mL at 48 hpf, and c) the probiotic combination at 18 hpf and V. coralliilyticus RE22 at 48 hpf. RNA was extracted from washed larvae after 72 hpf, and transcriptome sequencing was used to identify significant differentially expressed genes (DEGs) within each treatment. Larvae challenged with V. coralliilyticus showed enhanced expression of genes responsible for inhibiting immune signaling (i.e., TNFAIP3, PSMD10) and inducing apoptosis (i.e., CDIP53). However, when pre-treated with the probiotic combination, these genes were no longer differentially expressed relative to untreated control larvae. Additionally, pre-treatment with the probiotic combination increased expression of immune signaling proteins and immune effectors (i.e., IL-17, MyD88). Apparent immunomodulation in response to probiotic treatment corresponds to an increase in the survival of C. gigas larvae infected with V. coralliilyticus by up to 82%. These results indicate that infection with V. coralliilyticus can suppress the larval immune response while also prompting cell death. Furthermore, the results suggest that the probiotic combination treatment negates the deleterious effects of V. coralliilyticus on larval gene expression while stimulating the expression of genes involved in infection defense mechanisms.

Substantial gene expression shifts during larval transitions in the pearl oyster Pinctada margaritifera.

Early development stages in marine bivalve are critical periods where larvae transition from pelagic free-life to sessile mature individuals. The successive metamorphosis requires the expression of key genes, the functions of which might be under high selective pressure, hence understanding larval development represents key knowledge for both fundamental and applied research. Phenotypic larvae development is well known, but the underlying molecular mechanisms such as associated gene expression dynamic and molecular cross-talks remains poorly described for several nonmodel species, such as P. margaritifera. We designed a whole transcriptome RNA-sequencing analysis to describe such gene expression dynamics following four larval developmental stages: d-shape, Veliger, Umbo and Eye-spot. Larval gene expression and annotated functions drastically diverge. Metabolic function (gene expression related to lipid, amino acid and carbohydrate use) is highly upregulated in the first development stages, with increasing demand from d-shape to umbo. Morphogenesis and larval transition are partly ordered by Thyroid hormones and Wnt signaling. While larvae shells show some similar characteristic to adult shells, the cause of initialization of biomineralization differ from the one found in adults. The present study provides a global overview of Pinctada margaritifera larval stages transitioning through gene expression dynamics, molecular mechanisms and ontogeny of biomineralization, immune system, and sensory perception processes.

Associations between DNA methylation and gene regulation depend on chromatin accessibility during transgenerational plasticity

BackgroundEpigenetic processes are proposed to be a mechanism regulating gene expression during phenotypic plasticity. However, environmentally induced changes in DNA methylation exhibit little-to-no association with differential gene expression in metazoans at a transcriptome-wide level. It remains unexplored whether associations between environmentally induced differential methylation and expression are contingent upon other epigenomic processes such as chromatin accessibility. We quantified methylation and gene expression in larvae of the purple sea urchin Strongylocentrotus purpuratus exposed to different ecologically relevant conditions during gametogenesis (maternal conditioning) and modeled changes in gene expression and splicing resulting from maternal conditioning as functions of differential methylation, incorporating covariates for genomic features and chromatin accessibility. We detected significant interactions between differential methylation, chromatin accessibility, and genic feature type associated with differential expression and splicing.ResultsDifferential gene body methylation had significantly stronger effects on expression among genes with poorly accessible transcriptional start sites while baseline transcript abundance influenced the direction of this effect. Transcriptional responses to maternal conditioning were 4–13 × more likely when accounting for interactions between methylation and chromatin accessibility, demonstrating that the relationship between differential methylation and gene regulation is partially explained by chromatin state.ConclusionsDNA methylation likely possesses multiple associations with gene regulation during transgenerational plasticity in S. purpuratus and potentially other metazoans, but its effects are dependent on chromatin accessibility and underlying genic features.

Down-regulation of complement genes in lipopolysaccharide-challenged zebrafish (Danio rerio) larvae exposed to Indonesian propolis.

Although propolis has been reported for having anti-inflammatory activities, its effects on complement system has not been much studied. This research was conducted to find out the effects of Indonesian propolis on the expression levels of C3, C1r/s, Bf, MBL, and C6 in zebrafish larvae which were induced by lipopolysaccharide (LPS). Counting of macrophages migrating to yolk sac and liver histology were carried out. Larvae were divided into four groups: CON (cultured in E3 medium only), LPS (cultured in a medium containing 0.5 μg/L LPS), LPSIBU (cultured in a medium containing LPS, and then treated with 100 μg/L ibuprofen for 24 hours), and LPSPRO (cultured in a medium containing LPS, and then immersed in 14,000 μg/L propolis for 24 hours) groups. The results showed that complement gene expression in larvae from the LPSIBU and LPSPRO groups were generally lower than in larvae from the LPS group. The number of macrophage migrations to the yolk in the LPSPRO group was also lower than in the LPS group. Histological structure of liver in all groups were considered normal. This study shows that Indonesian propolis has the potential to be used as an alternative to the substitution of NSAIDs.

Effects of low salinities on growth, fatty acid composition, and transcriptome in Florida pompano (Trachinotus carolinus) at early developmental stages

The Florida pompano, Trachinotus carolinus, is a prime marine finfish candidate for commercial production in the United States (U.S.). Low salinity conditions (i.e., < 30 parts per thousand, ppt) can affect the early development of T. carolinus by increasing osmoregulatory stress. To address the knowledge gap regarding the impact of low salinity on Florida pompano health, we conducted an experiment to determine the optimal salinity for the culture of fingerlings. Larvae were reared in three salinities (10, 20, and 30 ppt) at local producers' farms and growth, fatty acid profiles, and transcriptome responses were quantified every three days after hatching (3, 6, 9, 12, 15, 18, and 24 days post hatch) and results were compared across salinities. Larvae reared at 10 ppt grew at a normal rate and the developmental stage had a greater impact than salinity on fatty acid composition and larval gene expression. Although there were few differentially expressed genes between salinities (n = 17), there was an upregulation of gene sets associated with ribosomes and oxidative phosphorylation and a downregulation of glycerophospholipid and carbon metabolism at lower salinities in comparison to higher salinities. In the early days of development, the fatty acids linoleic acid (LA), alpha linoleic acid (LNA), and 18:3 n-6 were proven to be important as potential energy fuels and precursors for long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, which was supported by the whole transcriptomics analysis. The gene expression of fatty acid synthesis genes coincided with changes in the fatty acid levels suggesting the ability of Florida pompano larvae to biosynthesize LC-PUFAs from LA and LNA precursors. Overall, this study suggests that it is possible to raise Florida pompano larvae for 24 DPH under low salinity conditions as low as 10 ppt. However, it is important to run this study for a longer period to investigate the long-term effects of low salinity on larval health and development. Importantly, this research serves as a model study for future on-farm collaborations and helps build a bridge between scientists and farmers in aquaculture.

Temperature and reduced pH regulate stress and biomineralization gene expression in larvae and post-larvae of the sand dollar Dendraster excentricus

ABSTRACT Seawater temperature, oxygen, salinity and pH are important abiotic factors, changes in which can generate stress in marine organisms. Subtidal and intertidal species, such as the sand dollar Dendraster excentricus, are daily exposed to stressors against which they have developed survival mechanisms to face environmental challenges. Analysing the expression of some key genes in response to stress factors due to changes in temperature and pH, especially in the early stages of development, opens a window of knowledge on the effect that these stressors have on benthic marine organisms. In the present work larvae and post-larvae of D. excentricus were exposed to high temperature and low pH scenarios. Survival, size and gene expression of five genes, involved in both stress response (hsp70 IV and hsp90 beta-like) and biomineralization for skeletogenesis (sm29, sm30A, carbonic anhydrase 14-like and mitochondrial proton/calcium exchanger protein LOC575637), were analysed in 4-, 6-, 8-arms, competent larvae and post-larvae. Survival of stressed larvae and post-larvae presented a significant decrease, up to 37% in some stages. A size reduction of almost 30 μm was observed when larvae were exposed to stressful conditions, except in competent larvae and in post-larvae where no significant changes were detected. After stress treatments, transcripts of hsp90 beta-like were up-regulated in all larval stages but hsp70 IV transcripts were not. Under tested stressful conditions sm29 and sm30A expression was down-regulated in larvae and post-larvae, while carbonic anhydrase 14-like and LOC575637 expressions were up-regulated. It is evident that tolerance to changes in seawater temperature and pH has a direct effect on metabolic functions of D. excentricus larvae and post-larvae, which depends on the developmental stage. If laboratory results are extrapolated to marine ecosystems, it is possible that populations of this structuring organism may be disturbed with subsequent damage to ecosystem balance, until resilient organisms acclimatize and adapt to their changing habitats.

Non-additive gene interactions underpin molecular and phenotypic responses in honey bee larvae exposed to imidacloprid and thymol

Understanding the cumulative risk of chemical mixtures at environmentally realistic concentrations is a key challenge in honey bee ecotoxicology. Ecotoxicogenomics, including transcriptomics, measures responses in individual organisms at the molecular level which can provide insights into the mechanisms underlying phenotypic responses induced by one or more stressors and link impacts on individuals to populations. Here, fifth instar honey bee larvae were sampled from a previously reported field experiment exploring the phenotypic impacts of environmentally realistic chronic exposures of the pesticide imidacloprid (5 μg.kg−1 for six weeks) and the acaricide thymol (250 g.kg−1 applied via Apiguard gel in-hive for four weeks), both separately and in combination. RNA-seq was used to discover individual and interactive chemical effects on larval gene expression and to uncover molecular mechanisms linked to reported adult and colony phenotypes. The separate and combined treatments had distinct gene expression profiles which represented differentially affected signaling and metabolic pathways. The molecular signature of the mixture was characterised by additive interactions in canonical stress responses associated with oxidative stress and detoxification, and non-additive interactions in secondary responses including developmental, neurological, and immune pathways. Novel emergent impacts on eye development genes correlated with long-term defects in visual learning performance as adults. This is consistent with these chemicals working through independent modes of action that combine to impact common downstream pathways, and highlights the importance of establishing mechanistic links between molecular and phenotypic responses when predicting effects of chemical mixtures on ecologically relevant population outcomes.

CYP4G8 is responsible for the synthesis of methyl-branched hydrocarbons in the polyphagous caterpillar of Helicoverpa armigera

Insect cuticular hydrocarbons (CHCs) have dual functions as physical barrier and chemical signals. The last step of CHC biosynthesis is known to be catalyzed by cytochrome P450 CYP4G in a number of insects. Until recently, studies on CYP4Gs in the context of functional evolution are rare. In this study, we analyzed sequence similarity and temporal-spatial expression patterns of the five CYP4G genes in the cotton bollworm Helicoverpa armigera, an important agricultural pest and also typical representative of lepidopteran insects. Moreover, the CRISPR/Cas9-induced knockout was used to clarify the roles of the five CYP4Gs in CHC biosynthesis. Temporal-spatial expression patterns revealed that CYP4G8 was highly expressed at all developmental stages and in most tissues examined. Larvae with CYP4G8 knocked out could not produce methyl-branched CHCs and failed to pupate, while larvae with the other four CYP4G genes knocked out (4G1-type-KO) showed no significant changes in their CHC profiles, weight gain and survival. Comparative transcriptomics revealed that knocking out CYP4G8 affected the global gene expression in larvae, especially down-regulated the expression of genes in the fatty acid biosynthetic pathway, while no significant change in 4G1-type-KO transcriptome was observed. These findings indicate that the five members of the CYP4G subfamily have undergone functional divergence: CYP4G8 maintains the essential function in CHC biosynthesis, while the function of the other four CYP4G genes remains unclear. Intriguingly, CYP4G8 has evolved to be a P450 enzyme responsible for the synthesis of larval methyl-branched hydrocarbons. The observation that CYP4G8 knockout is lethal strongly suggest that CYP4G8 may serve as a candidate target for the development of insecticidal agents for the control of cotton bollworms.

Comparative transcriptome analyses reveal changes of gene expression in larvae hatched from fresh and cryopreserved kelp grouper (Epinephelus moara) embryos

For the first time in fish, this study investigated the changes of gene expression in larvae hatched from fresh and cryopreserved embryos using RNA-seq. The transcriptome of kelp grouper from 1-day larvae hatched from fresh and cryopreserved embryos were obtained by high-throughput sequencing and analyzed by KEGG and GO database. The analysis results showed that 1815 unigenes were significantly differentially expressed (1165 up-regulated vs 650 down-regulated). Significantly down-regulated genes were enriched for eye development, cranial nerve development, sensory light stimulation and neurotransmitter transport terms, predicting that dehydration/rehydration or freezing/thawing during cryopreservation may affect development of the central nervous system, including the primordia of the eye. Moreover, many down-regulated genes were involved in calcium ion regulation, which may affect cell structure, as well as cell proliferation and differentiation. Meanwhile, gene expression of some proteins that protect neurons from the toxic effects of high concentrations of extracellular neurotransmitters significantly decreased, further suggesting that cryopreservation may have an effect on the central nervous system of larvae. In addition, huge amounts of primary data provided in present paper contribute to the genome and proteome databases of the grouper. These results will help other researchers better understand the effect of cryopreservation on fish embryos and improve exploration of the molecular mechanism of fish embryo cryoinjury.

Changes in Energy Reserves and Gene Expression Elicited by Freezing and Supercooling in the Antarctic Midge, Belgica antarctica.

Freeze-tolerance, or the ability to survive internal ice formation, is relatively rare among insects. Larvae of the Antarctic midge Belgica antarctica are freeze-tolerant year-round, but in dry environments, the larvae can remain supercooled (i.e., unfrozen) at subzero temperatures. In previous work with summer-acclimatized larvae, we showed that freezing is considerably more stressful than remaining supercooled. Here, these findings are extended by comparing survival, tissue damage, energetic costs, and stress gene expression in larvae that have undergone an artificial winter acclimation regime and are either frozen or supercooled at −5 °C. In contrast to summer larvae, winter larvae survive at −5 °C equally well for up to 14 days, whether frozen or supercooled, and there is no tissue damage at these conditions. In subsequent experiments, we measured energy stores and stress gene expression following cold exposure at −5 °C for either 24 h or 14 days, with and without a 12 h recovery period. We observed slight energetic costs to freezing, as frozen larvae tended to have lower glycogen stores across all groups. In addition, the abundance of two heat shock protein transcripts, hsp60 and hsp90, tended to be higher in frozen larvae, indicating higher levels of protein damage following freezing. Together, these results indicate a slight cost to being frozen relative to remaining supercooled, which may have implications for the selection of hibernacula and responses to climate change.

Patterns of gene expression in larvae of the giant grouper.

The giant grouper Epinephelus lanceolatus (Bloch 1790) is the largest bony fish found on coral reefs, achieving lengths of over 2 m. It has delicious flesh that is highly prized by the live reef fish trade. Consequently, declining wild populations have been listed as Vulnerable by IUCN. There is now aquaculture of E. lanceolatus but, as is often the case for marine fishes, larval feeding is a major production bottleneck. In particular, knowing how the digestive system develops and how this might determine the feed that larvae can digest to grow satisfactorily. In this issue, Anderson et al. (2018) used a transcriptomic approach to explore molecular processes that underpin development of appetite, feeding and digestion in E. lanceolatus larvae. They focussed on the first two weeks of life, a critical period for larval husbandry during which the larvae first open their mouth and start exogenous feeding. Because transcriptomics typically yield a very large quantity of information, the challenge is to reveal patterns. The authors used Self Organising Maps to hone in on genes that contribute to specific developmental milestones. They developed a conceptual model to show patterns of expression of transcripts in relation to major developmental stages, such as mouth-opening, onset of exogenous feeding, swim bladder development. There were clear trends for digestive enzyme transcripts, which tended to increase their expression in concert with mouth-opening and onset of exogenous feeding. Gene expression for various appetite-regulating factors was highly variable although, in some cases, this could be linked to important developmental milestones. Although such studies are inductive, they provide an essential knowledge base for development of targeted hypothesis-driven research. A major objective would be to find means of improving growth and survival of larvae without having to rely on feeding with copepods, which are expensive to produce in large quantities. Such improvements in methods for larval husbandry, in the farming of this highly-prized species, will take pressure off vulnerable wild stocks. The authors commented that Self Organising Maps can be applied to any project generating high volumes of multi-dimensional data, and are especially useful for time-course studies and pattern visualisation. As such, fish biologists are able to use this approach to more easily hone in on genes that contribute to specific developmental milestones, phenotype, mortality event, whose expression changes in response to particular stimuli.

The light at the end of the tunnel: photosensitivity in larvae of the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae)

AbstractInvestigations of light sensitivity and its physiological effects on insects developing within subcortical tree tissues are limited, presumably due to the assumption that cryptic microhabitats are completely devoid of light. In this study, we documented light-mediated behaviour and opsin gene expression in larvae of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae), an extremely important forest insect that is well adapted for development beneath the bark of pine (Pinus Linnaeus; Pinaceae) trees and is eyeless in the larval stage. Larvae were negatively phototactic, as they selected dark over light microhabitats in phototaxis assays. We recovered long-wavelength opsin transcripts from all life stages, including eggs and larvae, suggesting that D. ponderosae is photosensitive throughout its entire life cycle. Our results imply that photosensitivity contributes to immature D. ponderosae survival and that extraocular photoreception could be common among bark beetle larvae.

Transcriptional effects of polyethylene microplastics ingestion in developing zebrafish (Danio rerio)

Over the last few decades, plastic waste has become an increasing environmental concern as it accumulates in every environment on our planet. Though traditionally seen as a macroscopic problem (i.e., large plastic debris), plastic pollution is also evident at smaller scales. Indeed, the intentional industrial production of small plastic particles and the physical degradation of larger plastic debris have overtime resulted in an increased environmental prevalence of smaller plastic particles, including microplastics. While the effects of these small polymers on marine biota have been an important research focus, recent global surveys indicate that our freshwater lakes and rivers are also plagued by microplastics. However, despite these discoveries we currently have a limited understanding of the impact these particles may have on freshwater animals, particularly on vertebrate species. Thus, the aim of the present study was to assess the impact of high concentrations of microplastics (5 and 20 mg.L−1) on the early life stages in zebrafish, a model freshwater vertebrate model. To do this, we exposed embryonic and larval zebrafish to fluorescently labelled polyethylene microspheres for up to 14 days and assessed their microplastic content, growth, hatching and oxygen consumption rates. We then explored the molecular underpinnings of the microplastic response by RNA sequencing. Over the course of the exposure, we observed a consistent accumulation of microplastics in the gastrointestinal tract of the fish in a concentration dependent manner, but could not detect any detrimental effects of these particles on larval development, growth or metabolism. However, whole animal transcriptomics revealed that microplastics induced a transient and extensive change in larval gene expression within 48 h exposure, which largely disappeared by 14 days. However, as these transcriptional changes occurred during a critical period of larval development, we suggest that an evaluation of the potential long-term impact of these particles is warranted.

Development of the Astyanax mexicanus circadian clock and non-visual light responses

Most animals and plants live on the planet exposed to periods of rhythmic light and dark. As such, they have evolved endogenous circadian clocks to regulate their physiology rhythmically, and non-visual light detection mechanisms to set the clock to the environmental light-dark cycle. In the case of fish, circadian pacemakers are not only present in the majority of tissues and cells, but these tissues are themselves directly light-sensitive, expressing a wide range of opsin photopigments. This broad non-visual light sensitivity exists to set the clock, but also impacts a wide range of fundamental cell biological processes, such as DNA repair regulation. In this context, Astyanax mexicanus is a very intriguing model system with which to explore non-visual light detection and circadian clock function. Previous work has shown that surface fish possess the same directly light entrainable circadian clocks, described above. The same is true for cave strains of Astyanax in the laboratory, though no daily rhythms have been observed under natural dark conditions in Mexico. There are, however, clear alterations in the cave strain light response and changes to the circadian clock, with a difference in phase of peak gene expression and a reduction in amplitude. In this study, we expand these early observations by exploring the development of non-visual light sensitivity and clock function between surface and cave populations. When does the circadian pacemaker begin to oscillate during development, and are there differences between the various strains? Is the difference in acute light sensitivity, seen in adults, apparent from the earliest stages of development? Our results show that both cave and surface populations must experience daily light exposure to establish a larval gene expression rhythm. These oscillations begin early, around the third day of development in all strains, but gene expression rhythms show a significantly higher amplitude in surface fish larvae. In addition, the light induction of clock genes is developmentally delayed in cave populations. Zebrafish embryonic light sensitivity has been shown to be critical not only for clock entrainment, but also for transcriptional activation of DNA repair processes. Similar downstream transcriptional responses to light also occur in Astyanax. Interestingly, the establishment of the adult timing profile of clock gene expression takes several days to become apparent. This fact may provide mechanistic insight into the key differences between the cave and surface fish clock mechanisms.

Plasticity and evolutionary divergence in gene expression associated with alternative habitat use in larvae of the European Fire Salamander.

Transcriptomes of organisms reveal differentiation associated with the use of different habitats. However, this leaves open how much of the observed differentiation can be attributed to genetic differences or to transcriptional plasticity. In this study, we disentangle causes of differential gene expression in larvae of the European fire salamander from the Kottenforst forest in Germany. Larvae inhabit permanent streams and ephemeral ponds and represent an example of a young evolutionary split associated with contrasting ecological conditions. We hypothesized that adaptation towards differences in water temperature plays a role because the thermal regime between stream and pond habitats differs notably. Tissue samples from tail fins of larvae were collected to study gene expression using microarrays. We found ample evidence for differentiation among larvae occupying different habitats in nature with 2,800 of 11,797 genes being differentially expressed. We then quantified transcriptional plasticity towards temperature and genetic differentiation based on controlled temperature laboratory experiments. Gene-by-environment interactions modelling revealed that 28% of the gene expression divergence observed among samples in nature could be attributed to plasticity related to water temperature. Expression patterns of only a small number of 101 genes were affected by the genotype. Our analysis demonstrates that effects of environmental factors must be taken into account to explain variation of gene expression in salamanders in nature. Notwithstanding, it provides first evidence that genetic factors determined gene expression divergence between pond and stream ecotypes and could be involved in adaptive evolution.

Defensive repertoire of Drosophila larvae in response to toxic fungi.

Chemical warfare including insecticidal secondary metabolites is a well-known strategy for environmental microbes to monopolize a food source. Insects in turn have evolved behavioural and physiological defences to eradicate or neutralize the harmful microorganisms. We studied the defensive repertoire of insects in this interference competition by combining behavioural and developmental assays with whole-transcriptome time-series analysis. Confrontation with the toxic filamentous fungus Aspergillus nidulans severely reduced the survival of Drosophila melanogaster larvae. Nonetheless, the larvae did not behaviourally avoid the fungus, but aggregated at it. Confrontation with fungi strongly affected larval gene expression, including many genes involved in detoxification (e.g., CYP, GST and UGT genes) and the formation of the insect cuticle (e.g., Tweedle genes). The most strongly upregulated genes were several members of the insect-specific gene family Osiris, and CHK-kinase-like domains were over-represented. Immune responses were not activated, reflecting the competitive rather than pathogenic nature of the antagonistic interaction. While internal microbes are widely acknowledged as important, our study emphasizes the underappreciated role of environmental microbes as fierce competitors.

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae.

Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24°C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20–22°C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18°C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature.

Light regimes differentially affect baseline transcript abundance of stress-axis and (neuro)development-related genes in zebrafish (Danio rerio, Hamilton 1822) AB and TL larvae.

ABSTRACTMany strains of zebrafish (Danio rerio) are readily available. Earlier we observed differences between AB and Tupfel long-fin (TL) larvae regarding baseline hypothalamus-pituitary-interrenal (HPI) axis activity and (neuro)development. Light regimes, i.e. 14 h light:10 h dark and 24 h continuous dark or light, affect hatching rate and larval growth. Here, we assessed baseline transcript abundance of HPI-axis-related genes and (neuro)development-related genes of AB and TL larvae (5 days post fertilisation) using these light regimes. A principal component analysis revealed that in AB larvae the baseline expression of HPI-axis-related genes was higher the more hours of light, while the expression of (neuro)development-related genes was higher under 14 h light:10 h dark than under both continuous light or dark. In TL larvae, a complex pattern emerged regarding baseline expression of HPI-axis-related and (neuro)development-related genes. These data extend data of earlier studies by showing that light regimes affect gene-expression in larvae, and more importantly so, strengthen the notion of differences between larvae of the AB and TL strain. The latter finding adds to the growing database of phenotypical differences between zebrafish of the AB and TL strain.

Localization of Neuropeptide Gene Expression in Larvae of an Echinoderm, the Starfish Asterias rubens.

Neuropeptides are an ancient class of neuronal signaling molecules that regulate a variety of physiological and behavioral processes in animals. The life cycle of many animals includes a larval stage(s) that precedes metamorphic transition to a reproductively active adult stage but, with the exception of Drosophila melanogaster and other insects, research on neuropeptide signaling has hitherto largely focused on adult animals. However, recent advances in genome/transcriptome sequencing have facilitated investigation of neuropeptide expression/function in the larvae of protostomian (e.g., the annelid Platynereis dumerilii) and deuterostomian (e.g., the urochordate Ciona intestinalis) invertebrates. Accordingly, here we report the first multi-gene investigation of larval neuropeptide precursor expression in a species belonging to the phylum Echinodermata—the starfish Asterias rubens. Whole-mount mRNA in situ hybridization was used to visualize in bipinnaria and brachiolaria stage larvae the expression of eight neuropeptide precursors: L-type SALMFamide (S1), F-type SALMFamide (S2), vasopressin/oxytocin-type, NGFFYamide, thyrotropin-releasing hormone-type, gonadotropin-releasing hormone-type, calcitonin-type and corticotropin-releasing hormone-type. Expression of only three of the precursors (S1, S2, NGFFYamide) was observed in bipinnaria larvae but by the brachiolaria stage expression of all eight precursors was detected. An evolutionarily conserved feature of larval nervous systems is the apical organ and in starfish larvae this comprises the bilaterally symmetrical lateral ganglia, but only the S1 and S2 precursors were found to be expressed in these ganglia. A prominent feature of brachiolaria larvae is the attachment complex, comprising the brachia and adhesive disk, which mediates larval attachment to a substratum prior to metamorphosis. Interestingly, all of the neuropeptide precursors examined here are expressed in the attachment complex, with distinctive patterns of expression suggesting potential roles for neuropeptides in the attachment process. Lastly, expression of several neuropeptide precursors is associated with ciliary bands, suggesting potential roles for the neuropeptides derived from these precursors in control of larval locomotion and/or feeding. In conclusion, our findings provide novel perspectives on the evolution and development of neuropeptide signaling systems and neuroanatomical insights into neuropeptide function in echinoderm larvae.