Adaptation of the sulfophosphovanillin method of analysis of total lipids for various biological objects as exemplified by <i>Drosophila melanogaster</i>

Lipid metabolism is crucial in physiology. In recent decades the model object Drosophila melanogaster has been actively used in the study of the fundamental issues of lipid metabolism and its disorders, including obesity, as well as in the search for therapeutic goals for the treatment of metabolic disorders in humans. Quick and accurate quantification of lipid content is an important step in solving these problems. For the first time the method of quantitative measurement of total lipids with the use of the sulfophosphovanillin (SPV) method was described by Zöllner and colleagues in 1962, and adapted for insects by Van Handel on females of the yellow fever mosquito Aedes aegypti. The advantages of this method compared to traditional gravimetric and chromatographic methods of analysis are the use of a small amount of biological material, lack of need for complex manipulations with the sample, its high sensitivity, reproducibility and simplicity of implementation with a minimum set of equipment. Here, a modification of the Van Handel protocol is described, which allows the method to be adapted for quantitative determination of total lipids for various organisms as exemplified a widely used model, D. melanogaster. To test the effectiveness of the modified method, we measured the content of total lipids in D. melanogaster females carrying hypomorphic mutations of the dilp6 and dfoxo insulin signaling pathway genes compared to the wild-type Canton-S line, and showed that dilp6 took part in the regulation of fat metabolism, while dfoxo did not. The results obtained emphasize the effectiveness of the colorimetric method with the use of SPV reaction and spectrophotometry for the quantitative analysis of total lipids

The neuroendocrine stress-response in insects: the history of the development of the concept

The neuroendocrine stress-response is an effective defense mechanism against adverse influences of various nature. This reaction is universal and appears in response to stimuli that are unusual not just for living and habitat of the species, but also for each population. Here we review a progressive change of theoretical concepts, approaches and methods of research in this scientific field: beginning with the development of the stress concept by H. Selye and up to the present day. In 1982 H. Selye defined stress as a combination of stereotypical phylogenetic programmed  reactions of the organism that are caused by any strong, superstrong or extreme influences and are followed by a reorganization of the organism’s adaptive forces. The stress-causing agent was named a stressor. In the dy namics of the complex of nonspecific protectiveadaptive reactions that respond to a stressful influence aimed at cultivating the organism’s resistance to any factor, it is possible to logically identify three stages (“the Selye triad”): 1) alarm state, 2) resistance state, 3) exhaustion state. The duration and the expression of each stage can vary depending on the nature and strength of the stressor agent, the species of the animal and the physiological state of the organism. The lack of a hypothalamic-pituitary-adrenocortical system in insects was considered a proof of their inability to develop a stress reaction of the warm-blooded animals’ type. Nevertheless, since the early 1980s, enough evidence of the development of stress reaction in insects has been gathered, which emphasizes the conservative nature of the stress reaction in mammals and insects. The similarity in the neurochemical and physiological changes in invertebrates and vertebrates in response to a stressful influence indicates that the response to a stressor is a complex of ancient mechanisms preserved in evolution. Insects present unique opportunities for experimentation, which can allow us to understand the basic mechanisms of stress reactions. In insect larvae the mechanism of stress reaction has been studied in detail. In this century, the main efforts of researchers are aimed at studying the mechanisms of stress reaction in imago and genetic control of its individual links. The study of stress reaction in insects has both theoretical importance, as it demonstrates the convergence of evolutionary pathways of adaptive transformations in such distant taxa as insects and mammals, and practical importance, since the patterns of this reaction’s mechanisms can be used in modeling hereditary or acquired human diseases, in developing breeding methods for economically valuable insects and in finding ways to fight insect pest

Molecular mechanisms of autism as a form of synaptic dysfunction

Autism spectrum disorders are a separate group of defects with a very high genetic component. Genetic screening has identified hundreds of mutations and other genetic variations associated with autism, and bioinformatic analysis of signaling pathways and gene networks has led to understanding that many of these mutational changes are involved in the functioning of synapses. A synapse is a site of electrochemical communication between neurons and an essential subunit for learning and memory. Interneuronal communicative relationships are plastic. The most prominent forms of synaptic plasticity are accompanied by changes in protein biosynthesis, both in neuron body and in dendrites. Protein biosynthesis or translation is a carefully regulated process, with a central role played by mTOR (mammalian or mechanistic target of rapamycin). Normally mTOR-regulated translation is slightly inhibited, and in most cases mutational damage to at least one of the links of the mTOR signaling pathway, increases translation and leads to impaired synaptic plasticity and behavior. Deregulation of the local translation in dendrites is connected with the following monogenic autism spectrum disorders: neurofibromatosis type 1, Noonan syndrome, Costello syndrome, Cowden syndrome, tuberous sclerosis, fragile X chromosome, syndrome, and Rett syndrome. The review considers the most important mutations leading to monogenic autism, as well as the possibility of a mechanism-based treatment of certain disorders of the autism spectrum

Comparative analysis of the ftness of Drosophila virilis lines contrasting in response to stress

One of the crucial elements contributing to the adaptation of organisms to unfavorable environmental conditions is the reaction of stress. The study of its genetic control and role in adaptation to unfavorable conditions are of special interest. The juvenile hormone (JH) acts as a gonadotropic hormone in adult insects controlling the development of the ovaries, inducing vitellogenesis and oviposition. It was shown that a decrease in JH degradation in individuals reacting to adverse conditions by stress reaction (R­individuals) causes delay in egg laying and seems to allow the population to “wait out” the unfavorable conditions, thereby contributing to the adaptation at the population level. However, monitoring natural populations of D. melanogaster for the capability of stress reaction demonstrated that they have a high percentage of individuals incapable of it (NR­individuals). The study of reproductive characteristics of R­ and NR­individuals showed that under normal conditions R­individuals have the advantage of procreating offspring. Under unfavorable conditions, if the stressor is intense enough, NR­individuals die, but if its intensity is low, then they, unlike R­individuals, continue to produce offspring. Based on these data, it was hypothesized that the balance of R­ and NR­alleles in the population ensures its adaptation under frequent stresses of low intensity. To verify the hypothesis by an experiment, the ftness characteristics (lifespan, fecundity) of the R and NR lines of D. virilis were studied under normal conditions and under regular heat stress of various frequency

The transcription factor dFOXO controls the expression of insulin pathway genes and lipids content under heat stress in <i>Drosophila melanogaster</i>

The insulin/insulin-like growth factor signaling (IIS) pathway is one of the key elements in an organism’s response to unfavourable conditions. The deep homology of this pathway and its evolutionary conservative role in controlling the carbohydrate and lipid metabolism make it possible to use Drosophila melanogaster for studying its functioning. To identify the properties of interaction of two key IIS pathway components under heat stress in D. melanogaster (the forkhead box O transcription factor (dFOXO) and insulin-like peptide 6 (DILP6), which intermediates the dFOXO signal sent from the fat body to the insulin-producing cells of the brain where DILPs1–5 are synthesized), we analysed the expression of the genes dilp6, dfoxo and insulin-like receptor gene (dInR) in females of strains carrying the hypomorphic mutation dilp641 and hypofunctional mutation foxoBG01018. We found that neither mutation influenced dfoxo expression and its uprise under short-term heat stress, but both of them disrupted the stress response of the dilp6 and dInR genes. To reveal the role of identified disruptions in metabolism control and feeding behaviour, we analysed the effect of the dilp641 and foxoBG01018 mutations on total lipids content and capillary feeding intensity in imago under normal conditions and under short-term heat stress. Both mutations caused an increase in these parameters under normal conditions and prevented decrease in total lipids content following heat stress observed in the control strain. In mutants, feeding intensity was increased under normal conditions; and decreased following short-term heat stress in all studied strains for the first 24 h of observation, and in dilp641 strain, for 48 h. Thus, we may conclude that dFOXO takes part in regulating the IIS pathway response to heat stress as well as the changes in lipids content caused by heat stress, and this regulation is mediated by DILP6. At the same time, the feeding behaviour of imago might be controlled by dFOXO and DILP6 under normal conditions, but not under heat stress

Differentially expressed profiles in the larval testes of Wolbachia infected and uninfected Drosophila

BACKGROUND: Wolbachia are endosymbiotic bacteria that are frequently found in arthropods and nematodes. These maternally inherited bacteria manipulate host reproduction by several mechanisms including cytoplasmic incompatibility (CI). CI is the most common phenotype induced by Wolbachia and results in the developmental arrest of embryos derived from crosses between Wolbachia-infected males and uninfected females. Although the molecular mechanisms of CI are currently unknown, several studies suggest that host sperm is modified by Wolbachia during spermatogenesis. RESULTS: We compared the gene expression of Drosophila melanogaster larval testes with and without the wMel strain of Wolbachia to identify candidate genes that could be involved in the interaction between Wolbachia and the insect host. Microarray, quantitative RT-PCR and in situ hybridization analyses were carried out on D. melanogaster larval testes to determine the effect of Wolbachia infection on host gene expression. A total of 296 genes were identified by microarray analysis to have at least a 1.5 fold change [q-value < 5%] in expression. When comparing Wolbachia-infected flies to uninfected flies, 167 genes were up-regulated and 129 genes down-regulated. Differential expression of genes related to metabolism, immunity, reproduction and other functions were observed. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes are differentially expressed in the testes of the 3rd instar larvae of Wolbachia-infected and uninfected flies. In situ hybridization demonstrated that Wolbachia infection changes the expression of several genes putatively associated with spermatogenesis including JH induced protein-26 and Mst84Db, or involved in immune (kenny) or metabolism (CG4988-RA). CONCLUSIONS: Wolbachia change the gene expression of 296 genes in the larval testes of D. melanogaster including genes related to metabolism, immunity and reproduction. Interestingly, most of the genes putatively involved in immunity were up-regulated in the presence of Wolbachia. In contrast, most of the genes putatively associated with reproduction (especially spermatogenesis) were down-regulated in the presence of Wolbachia. These results suggest Wolbachia may activate the immune pathway but inhibit spermatogenesis. Our data provide a significant panel of candidate genes that may be involved in the interaction between Wolbachia and their insect hosts. This forms a basis to help elucidate the underlying mechanisms of Wolbachia-induced CI in Drosophila and the influence of Wolbachia on spermatogenesis

Transcriptome profiling of ontogeny in the acridid grasshopper Chorthippus biguttulus

Acridid grasshoppers (Orthoptera:Acrididae) are widely used model organisms for developmental, evolutionary, and neurobiological research. Although there has been recent influx of orthopteran transcriptomic resources, many use pooled ontogenetic stages obscuring information about changes in gene expression during development. Here we developed a de novo transcriptome spanning 7 stages in the life cycle of the acridid grasshopper Chorthippus biguttulus. Samples from different stages encompassing embryonic development through adults were used for transcriptomic profiling, revealing patterns of differential gene expression that highlight processes in the different life stages. These patterns were validated with semi-quantitative RT-PCR. Embryonic development showed a strongly differentiated expression pattern compared to all of the other stages and genes upregulated in this stage were involved in signaling, cellular differentiation, and organ development. Our study is one of the first to examine gene expression during post-embryonic development in a hemimetabolous insect and we found that only the fourth and fifth instars had clusters of genes upregulated during these stages. These genes are involved in various processes ranging from synthesis of biogenic amines to chitin binding. These observations indicate that post-embryonic ontogeny is not a continuous process and that some instars are differentiated. Finally, genes upregulated in the imago were generally involved in aging and immunity. Our study highlights the importance of looking at ontogeny as a whole and indicates promising directions for future research in orthopteran development

Fine-Scale Mapping of Natural Variation in Fly Fecundity Identifies Neuronal Domain of Expression and Function of an Aquaporin

To gain insight into the molecular genetic basis of standing variation in fitness related traits, we identify a novel factor that regulates the molecular and physiological basis of natural variation in female Drosophila melanogaster fecundity. Genetic variation in female fecundity in flies derived from a wild orchard population is heritable and largely independent of other measured life history traits. We map a portion of this variation to a single QTL and then use deficiency mapping to further refine this QTL to 5 candidate genes. Ubiquitous expression of RNAi against only one of these genes, an aquaporin encoded by Drip, reduces fecundity. Within our mapping population Drip mRNA level in the head, but not other tissues, is positively correlated with fecundity. We localize Drip expression to a small population of corazonin producing neurons located in the dorsolateral posterior compartments of the protocerebrum. Expression of Drip–RNAi using both the pan-neuronal ELAV-Gal4 and the Crz-Gal4 drivers reduces fecundity. Low-fecundity RILs have decreased Crz expression and increased expression of pale, the enzyme encoding the rate-limiting step in the production of dopamine, a modulator of insect life histories. Taken together these data suggest that natural variation in Drip expression in the corazonin producing neurons contributes to standing variation in fitness by altering the concentration of two neurohormones

A Link between Atmospheric Pressure and Fertility of Drosophila Laboratory Strains

Standardization of conditions under which insects are kept is of great importance when studying their physiology and researchers do their best to maintain it. Nevertheless, sometimes an obvious side effect of some unaccounted factor affecting insects’ reproduction can be revealed even under thoroughly controlled laboratory conditions. We faced such a phenomenon when studying the fertility level in two wild type Drosophila melanogaster strains. For fertility analysis, 50 newly emerged females and 50 males of each strain under study were transferred to fresh medium daily within 10 days. We found out that fertility of both strains was stable on days 2–10 after the oviposition onset in one experiment, while in another one it was significantly decreased during days 5–10. When compared to publicly available meteorological data, these changes in the fertility level demonstrated a strong association with one weather factor: barometric pressure. Thus, we conclude that changes in atmospheric pressure can be considered a factor affecting insects reproduction and discuss a possible mechanism of their influence on fertility