rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila

Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgn^G4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgn^G4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgn^G4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila.     

Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.     

Biosynthesis of contact sex pheromone in the female tsetse fly, Glossina morsitans morsitans Westwood

Injection of [2,3 ¹⁴C] sodium succinate into recently emerged, unfed females of Glossina morsitans morsitans resulted in incorporation of radiolabel mainly into surface cuticular alkanes. In vivo experiments with intact flies showed that the distribution of labelled alkanes depended on fly mobility, the legs of unrestrained flies possessing proportionately larger amounts of radioactive hydrocarbon material than those of flies whose legs were tied together with a silk suture. The heads of both restrained and unrestrained flies contained proportionately more material per unit surface area than did any other body part. However, ablation experiments and in vitro incubation showed that the most active incorporation of label into alkanes occurred in the abdomen and that all dorsal abdominal segments were equally active. The ventral abdomen also incorporated label into cuticular alkanes in vitro, but other body parts were apparently less able to do so. The sex pheromone of G. m. morsitans is a trimethyl-substituted alkane, the labelling of which appeared to be in proportion to the relative abundance of its methyl groups among those of the other alkanes of the cuticle following injection of either intact or legless flies. Hence it is proposed that sex pheromone is synthesized along with other cuticular alkanes mainly by cells closely associated with the abdominal cuticle of females and that it is spread over the external surface both by diffusion and by grooming which leads to accumulations of hydrocarbon material on the legs.     

Taste responses of Drosophila melanogaster

The amounts of sugar solution consumed by Drosophila melanogaster flies were determined. Starved and desiccated flies of a wild type strain (QA) consume 7?9 × 10^?2 λ of a 0.3 M sucrose solution per fly during the first hour and less later. They consume more of the 0.3 M sucrose solution than of the more diluted and the more concentrated solutions. In preference-aversion tests the flies discriminated between water and various sugar solutions, and between different sugar concentrations. Contrary to other fly species these flies did not prefer 0.05 M fructose over 0.05 M glucose. 0.3–0.5 M NaCl added to 0.1 M sucrose turned a preference over 0.01 sucrose into an aversion. A mutant, Lot-94, selected for its increased consumption of a 1 M NaCl solution was found to consume more of all test solutions. The amount of NaCl that had to be added to 0.1 M sucrose to turn the preference over 0.01 M sucrose by the mutant flies into aversion was not different from that found for the wild type flies.     

Regulation of cuticular hydrocarbon profile maturation by Drosophila tanning hormone,bursicon, and its interaction with desaturase activity

Shortly after emergence the exoskeleton (cuticle) of adult insects is rapidly expanded, hardened (sclerotized), and pigmented (melanized). In parallel with this process, the oenocytes, which are large polyploid cells located below the abdominal epidermis, secrete onto the cuticle a cocktail of cuticular hydrocarbons (CHs) and waxes. These improve the waterproofing of the cuticle, and also provide important chemosensory and pheromonal cues linked with gender, age, and species differentiation. The hardening and pigmentation of the new cuticle are controlled by the neurohormone, bursicon, and its receptor, encoded by the DLGR2 receptor, rickets (rk); by contrast, little is known about the timecourse of changes in CH profile and about the role of bursicon in this process. Here we show in Drosophila that rk function is also required for the normal maturation of the fly's CH profile, with flies mutant for rk function showing dramatically elevated levels of CHs. Interestingly, this effect is mostly abrogated by mutations in the Δ9 desaturase encoded by the desaturase1 gene, which introduces a first double bond into elongated fatty-acid chains, suggesting that desaturase1 acts downstream of rk. In addition, flies mutant for rk showed changes in the absolute and relative levels of specific 7-monoenes (in males) and 7,11-dienes (in females). The fact that these differences in CH amounts were obtained using extractions of very different durations suggests that the particular CH profile of flies mutant for rk is not simply due to their unsclerotized cuticle but that bursicon may be involved in the process of CH biosynthesis itself.     

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.     

The biology of the black larval mutant of the tobacco hornworm, Manduca sexta

A mutant of the tobacco hornworm, Manduca sexta, was found to form black melanized cuticle in the last larval instar. This black phenotype is due to a single sex-linked gene whose expression can be changed by one or more modifier genes. The expression of the mutant phenotype is prevented by juvenile hormone (JH) application at the time of head cap apolysis during the moulting cycle to the last larval instar. The bl mutant is equally as sensitive to JH at this time as is a neck-ligated wild type larva, ruling out an absence of hormone receptors or a difference in JH metabolism. The bl corpora allata were found to be less active at this time than were those of the wild type larva, suggesting that the defect resides in the control of the corpora allata. Since selection for complete expression of the bl phenotype is easy, this mutant provides the basis for an ultrasensitive JH bioassay to be described in a forthcoming paper.     

The relationship between locomotor activity and sexual behaviour in ebony strains of Drosophila melanogaster

Male flies homozygous for the ebony¹¹ mutant allele suffer a severe visual impairment and consequently show poor mating success. However, ebony males show improved mating in darkness because they initiate courtship more quickly than in the light. They also exhibit a competitive superiority over wild-type males under darkened conditoons that correlates with the observed increase in the mutant's locomotor activity. Locomotor activity levels were subsequently shown to correlate with several indices of mating success under both dark and illuminated conditions. The evolutionary implications of this relationship between the two characters are discussed.     

Defective gut function in drop-dead mutant Drosophila

Mutation of the gene drop-dead (drd) causes adult Drosophila to die within 2 weeks of eclosion and is associated with reduced rates of defecation and increased volumes of crop contents. In the current study, we demonstrate that flies carrying the strong allele drdlwf display a reduction in the transfer of ingested food from the crop to the midgut, as measured both as a change in the steady-state distribution of food within the gut and also in the rates of crop emptying and midgut filling following a single meal. Mutant flies have abnormal triglyceride (TG) and glycogen stores over the first 4 days post-eclosion, consistent with their inability to move food into the midgut for digestion and nutrient absorption. However, the lifespan of mutants was dependent upon food presence and quality, suggesting that at least some individual flies were able to digest some food. Finally, spontaneous motility of the crop was abnormal in drdlwf flies, with the crops of mutant flies contracting significantly more rapidly than those of heterozygous controls. We therefore hypothesize that mutation of drd causes a structural or regulatory defect that inhibits the entry of food into the midgut.     

A comparative study of specific chromatic adaptation of the wildtype and trp mutant of Drosophila melanogaster

The trp mutant of Drosophila melanogaster was re-examined and compared with the wildtype using monochromatic blue and orange light to manipulate the bi-stable visual pigment states in the peripheral retinula cells R1-6 of white-eyed flies. Recovery of sensitivity by application of orange light either during or after blue-adaptation is different in w;trp flies from that in bw;cn flies and does not proceed as predicted from the trp genotype. Blue-adaptation by isolating the activity of the central retinula cells confirms that the trp lesion affects these receptors also.     

Water loss and respiration of Lucilia cuprina during development within the puparium

The rate of loss of water and the rate of uptake of oxygen were measured continuously throughout the development of Lucilia cuprina within the puparium. Changes in these parameters were correlated with changes observed in morphology of cuticles and respiratory structures during development.In development at 26°C, there is, at 20–22 hr after puparium formation a major loss of water by mechanical expulsion of moulting fluid chiefly through the posterior larval spiracles after the severing of the posterior larval tracheae. This loss of water is essential to survival and is followed by an extremely low rate of water loss attributed to slow diffusion of water through the resulting air gap between the pupal cuticle and the puparium. There is an increase in oxygen consumption during the pupal movements associated with the casting of the larval tracheae followed by a sharp reduction in oxygen consumption until the pupal horns are everted a short time later. This combination of physiological events enables development to proceed over a wide range of conditions in the puparial environment.     

Effects of the eyefluke, Diplostomum spathaceum, on the behaviour of dace (Leuciscus leuciscus)

The behaviour of dace infected with Diplostomum spathaceum was investigated in the laboratory. As the number of parasites present in the eye increased, the efficiency with which the fish fed on Gammarus pulex declined. The loss of efficiency was compensated for by an increase in the time devoted to feeding. Heavily infected fish spent more time in the surface layers of the water. This may increase the likelihood that a gull might eat the fish and thereby continue the life-cycle of the parasite. The parasite modifies the behaviour of its host in a way that increases the parasite's chances of survival at the expense of the host.     

Reversion of Scenedesmus photosynthetic mutants

The chlorophyll-protein complex associated with Photosystem I, CP-I, which is absent in Scenedesmus obliquus mutant 8 grown in the dark, is present in light-grown photosynthetically competent cultures of Scenedesmus 8, contrary to a previously published report (Gee, R., Saltman, P. and Weaver, E. (1969) Biochim. Biophys. Acta 189, 106–115). This change from mutant to wild-type traits is not due to photo-adaptation, but reflects the genetic reversion of some of the cells in the dark-grown population, as shown by the following evidence. (1) Individual cultures take different lengths of time to regain competence, whether started from the same or different dark-grown mutant 8 cultures. (2) Competent cells do not de-adapt when returned to the dark. (3) The appearance of wild-type traits is gradual. (4) A small number of cells in mutant populations are wild-type and are selected for in the light. The reversion rate of mutant 8 to the wild-type is high compared to that of mutant 11.     

Metamorphic changes of wild type and mutant Drosophila tissues induced by 20-hydroxy ecdysone in vitro

Wild type (Oregon R) and non-pupariating as well as late-pupariating mutant larval tissues were cultured in vitro up to 5 weeks with and without 20-hydroxy ecdysone (1 μg/ml). The following responses were elicited by the hormone: in the case of wild type tissues detachment of the larval epidermis and muscles from the cuticle; puparial tanning and sclerotization of the larval cuticle; dissociation of the fat body into single cells; inhibition of the movement of the hind intestine. Most of these responses developed within 1 week of culturing. Of the 4 mutants tested, 3 behaved like the wild type. In cultures of ?(1)npr-1, however, puparial tanning, disc evagination, and inhibition of the movement of the hind intestine was abnormally weak and the dissociation of fat body was not observed at all. Detachment of the epidermis and muscles as well as formation of the pupal cuticle by disc tissue occurred normally. The results are discussed with respect to the ecdysteroid-induced metamorphosis of the tissues and the autonomy of mutant gene action.     

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.     

Fine-scale genetic breaks driven by historical range dynamics and ongoing density-barrier effects in the estuarine seaweed Fucus ceranoides L.

Background

In the laboratory, the Drosophila melanogaster heat shock protein Hsp90 can buffer the phenotypic effects of genetic variation. Laboratory experiments either manipulate Hsp90 activity pharmacologically, or they induce mutations with strong effects in the gene Hsp83, the single-copy fly gene encoding Hsp90. It is unknown whether observations from such laboratory experiments are relevant in the wild.

Results

We here study naturally occurring mutations in Hsp83, and their effects on fitness and phenotypic buffering in flies derived from wild populations. We examined more than 4500 flies from 42 Drosophila populations distributed world-wide for insertions or deletions of mobile DNA in or near the Hsp83 gene. The insertions we observed occur at low population frequencies, and reduce Hsp83 gene expression. In competition experiments, mutant flies performed much more poorly than wild-type flies. Mutant flies were also significantly less fecund and shorter-lived than wild-type flies, as well as less well buffered against cryptic deleterious variation, as we show through inbreeding experiments. Specifically, in Hsp83 mutant flies female fecundity dropped to much lower levels after inbreeding than in wild-type flies. At even slightly elevated temperatures, inbred mutant Hsp83 populations went extinct, whereas inbred wild-type populations persisted.

Conclusions

Our work shows that Hsp90, a regulator of the stress response and of signaling, helps buffer deleterious variation in fruit flies derived from wild population, and that its buffering role becomes even more important under heat stress.