A gene-driven recovery mechanism: Drosophila larvae increase feeding activity for post-stress weight recovery.

Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post-stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non-typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up-regulation of post-stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post-stress periods.

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress.

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

Function of desiccate in gustatory sensilla of drosophila melanogaster.

Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

Switching between humoral and cellular immune responses in Drosophila is guided by the cytokine GBP.

Insects combat infection through carefully measured cellular (for example, phagocytosis) and humoral (for example, secretion of antimicrobial peptides (AMPs)) innate immune responses. Little is known concerning how these different defense mechanisms are coordinated. Here, we use insect plasmatocytes and hemocyte-like Drosophila S2 cells to characterize mechanisms of immunity that operate in the haemocoel. We demonstrate that a Drosophila cytokine, growth-blocking peptides (GBP), acts through the phospholipase C (PLC)/Ca(2+) signalling cascade to mediate the secretion of Pvf, a ligand for platelet-derived growth factor- and vascular endothelial growth factor-receptor (Pvr) homologue. Activated Pvr recruits extracellular signal-regulated protein kinase to inhibit humoral immune responses, while stimulating cell 'spreading', an initiating event in cellular immunity. The double-stranded RNA (dsRNA)-targeted knockdown of either Pvf2 or Pvr inhibits GBP-mediated cell spreading and activates AMP expression. Conversely, Pvf2 overexpression enhances cell spreading but inhibits AMP expression. Thus, we describe mechanisms to initiate immune programs that are either humoral or cellular in nature, but not both; such immunophysiological polarization may minimize homeostatic imbalance during infection.

Immunoevasive protein (IEP)-containing surface layer covering polydnavirus particles is essential for viral infection.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp.

Gain of long tonic immobility behavioral trait causes the red flour beetle to reduce anti-stress capacity.

Tonic immobility (death-feigning) behavior of the red flour beetle, Tribolium castaneum, is a predator defense mechanism; it is a reflex elicited when a beetle is jarred with the substrate, often a result of the activities of a predator. We previously demonstrated that the frequency of predation by a jumping spider, Hasarius adansoni, was significantly lower among beetles with higher frequencies and longer durations of tonic immobility (L-type) than those with lower frequencies and shorter durations of tonic immobility (S-type). However, we found that the population of L-type beetles is much smaller than that of S-type beetles in their natural habitat. Here we demonstrated that L-type beetles are significantly more sensitive to environmental stressors such as mechanical vibration and high or low temperatures. We measured expression levels of stress-responsive genes such as heat shock proteins (Hsps) and antioxidant enzymes in both types of beetles. Among the genes we investigated, only catalase gene expression levels were significantly higher in S-type than in L-type beetles. Furthermore, a similar difference in the gene expression was observed in the T. castaneum ortholog of the insect cytokine growth-blocking peptide (GBP) gene. These results indicate the possibility that high expression of catalase and GBP in S-type beetles contributes to augmentation of their anti-stress capacity and expansion of their population in their natural habitat.

Gustatory sensing mechanism coding for multiple oviposition stimulants in the swallowtail butterfly, Papilio xuthus.

The swallowtail butterfly, Papilio xuthus, selectively uses a limited number of plants in the Rutaceae family. The butterfly detects oviposition stimulants in leaves through foreleg chemosensilla and requires a specific combination of multiple oviposition stimulants to lay eggs on the leaf of its host plants. In this study, we sought to elucidate the mechanism underlying the regulation of oviposition behavior by multiple oviposition stimulants. We classified chemosensilla on the tarsomere of the foreleg into three types (L1, L2, and S) according to their size and response to oviposition stimulants and general tastants. The L1 was more abundant in females than in males and responded preferentially to oviposition stimulants. Both L2 and S were common to both sexes and responded to general tastants. We found that five oviposition stimulants (synephrine, stachydrine, 5-hydroxy-Nω-methyltryptamine, narirutin, and chiro-inositol) elicited spikes from three specific gustatory receptor neurons (GRNs) within L1 sensilla. These three GRNs responded to a mixture of the five stimulants at concentrations equivalent to those found in the whole-leaf extract of citrus, and the mixture induced oviposition at levels comparable to whole-leaf extract. We propose that oviposition is triggered by the firing of three specific GRNs in L1 sensilla that encode the chemical signatures of multiple oviposition stimulants.

A gustatory receptor involved in host plant recognition for oviposition of a swallowtail butterfly.

Swallowtail butterflies belonging to the family of Papilionidae selectively utilize a limited number of plants from a single or a few families. Female butterflies lay eggs on their host only when they detect specific chemicals through their foreleg chemosensilla while drumming on the leaf surface. Here we show that the butterfly, Papilio xuthus, uses a gustatory receptor specific for synephrine to select its host in oviposition behaviour. We identify a gustatory receptor gene involved in the recognition of an oviposition stimulant, synephrine, from the P. xuthus by a combination of in silico, in vitro and in vivo approaches. The receptor, PxutGr1, responds specifically to synephrine in Sf9 cells. The sensitivity of tarsal taste sensilla to synephrine and the oviposition behaviour in response to synephrine are strongly reduced after injecting double-stranded RNA of PxutGr1 into pupae. These observations indicate that the receptor PxutGr1 represents a key factor in host specialization in P. xuthus.

Identification of a novel gene, anorexia, regulating feeding activity via insulin signaling in Drosophila melanogaster.

Feeding activities of animals, including insects, are influenced by various signals from the external environment, internal energy status, and physiological conditions. Full understanding of how such signals are integrated to regulate feeding activities has, however, been hampered by a lack of knowledge about the genes involved. Here, we identified an anorexic Drosophila melanogaster mutant (GS1189) in which the expression of a newly identified gene, Anorexia (Anox), is mutated. In Drosophila larvae, Anox encodes an acyl-CoA binding protein with an ankyrin repeat domain that is expressed in the cephalic chemosensory organs and various neurons in the central nervous system (CNS). Loss of its expression or disturbance of neural transmission in Anox-expressing cells decreased feeding activity. Conversely, overexpression of Anox in the CNS increased food intake. We further found that Anox regulates expression of the insulin receptor gene (dInR); overexpression and knockdown of Anox in the CNS, respectively, elevated and repressed dInR expression, which altered larval feeding activity in parallel with Anox expression levels. Anox mutant adults also showed significant repression of sugar-induced nerve responses and feeding potencies. Although Anox expression levels did not depend on the fasting and feeding states cycle, stressors such as high temperature and desiccation significantly repressed its expression levels. These results strongly suggest that Anox is essential for gustatory sensation and food intake of Drosophila through regulation of the insulin signaling activity that is directly regulated by internal nutrition status. Therefore, the mutant strain lacking Anox expression cannot enhance feeding potencies even under starvation.

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster.

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.

Analysis of hunger-driven gene expression in the Drosophila melanogaster larval central nervous system.

A transposon-inserted mutant of Drosophila melanogaster was recently identified, and the larvae show no food preference (Ryuda and Hayakawa, 2005). To reveal the genetic mechanism underlying the preference change in this mutant, a large-scale oligo-DNA microarray screening was carried out to identify genes whose expression is different in control and mutant strains. We focused especially on hunger-driven changes in gene expression in the larval central nervous system (CNS) of both strains, because the state of food depletion should promote a feeding response due to changed expression of certain genes in the CNS. We identified 22 genes whose expression changed after starvation in either or both of the two strains. Quantitative RT-PCR analyses confirmed the expression changes in four genes, CG6271, CG6277, CG7953, and new glue 3 (ng3, encoding a putative structural molecule). CG6271 and CG6277 encode triacylglycerol lipase, and CG7953 produces a protein homologous to a juvenile hormone (JH) binding protein. The expression of these two groups of genes was enhanced in control strain larvae with a normal food preference but not in GS1189 strain larvae. Given that these genes contribute to mediating hunger-driven changes in food preference and intake in D. melanogaster larvae, the dysfunction of these key genes could cause the defect in food preference observed in GS1189-strain larvae.

A gene involved in the food preferences of larval Drosophila melanogaster.

To examine the mechanism by which insects change their food preferences, a simple method was developed to measure their preferences. By using this method, we demonstrated preference of Drosophila melanogaster larvae of the yw control strain for a food based on soybeans over one based on cornmeal. We then screened for mutant strains with food preferences clearly different from the control yw strain, using the Gene Search collection of P-element insertions (GS strains). Among 380 GS strains screened using an assay plate-containing soybean and corn tastants, we identified one mutant, GS1189 that did not show any preference for either of the foods. Further behavioral assays indicated that the GS1189 larvae could have impaired olfactory and gustatory systems. The fact that the CG33071 gene expression was inactivated by the P-element insertion in the GS1189 strain, and that reversion of this gene completely recovered the normal food preference, indicates that this gene contributes to the control of food preferences in Drosophila larvae.