Pigment-dispersing factor in the locust abdominal ganglia may have roles as circulating neurohormone and central neuromodulator

Pigment-dispersing factor (PDF) is a neuropeptide that has been indicated as a likely output signal from the circadian clock neurons in the brain of Drosophila. In addition to these brain neurons, there are PDF-immunoreactive (PDFI) neurons in the abdominal ganglia of Drosophila and other insects; the function of these neurons is not known. We have analyzed PDFI neurons in the abdominal ganglia of the locust Locusta migratoria. These PDFI neurons can first be detected at about 45% embryonic development and have an adult appearance at about 80%. In each of the abdominal ganglia (A3-A7) there is one pair of lateral PDFI neurons and in each of the A5-A7 ganglia there is additionally a pair of median neurons. The lateral neurons supply varicose branches to neurohemal areas of the lateral heart nerves and perisympathetic organs, whereas the median cells form processes in the terminal abdominal ganglion and supply terminals on the hindgut. Because PDF does not influence hindgut contractility, it is possible that also these median neurons release PDF into the circulation. Release from one or both the PDFI neuron types was confirmed by measurements of PDF-immunoreactivity in hemolymph by enzyme immunoassay. PDF applied to the terminal abdominal ganglion triggers firing of action potentials in motoneurons with axons in the genital nerves of males and the 8th ventral nerve of females. Because this action is blocked in calcium-free saline, it is likely that PDF acts via interneurons. Thus, PDF seems to have a modulatory role in central neuronal circuits of the terminal abdominal ganglion that control muscles of genital organs.     

Pigment‐dispersing factor signaling in the circadian system of Caenorhabditis elegans

The neuropeptide pigment‐dispersing factor (PDF) is important for the generation and entrainment of circadian rhythms in the fruitfly Drosophila melanogaster. Recently two pdf homologs, pdf‐1 and pdf‐2, and a PDF receptor, pdfr‐1, have been found in Caenorhabditis elegans and have been implicated in locomotor activity. In this work, we have studied the role of the PDF neuropeptide in the circadian system of C. elegans and found that both pdf‐1 and pdf‐2 mutants affect the normal locomotor activity outputs. In particular, loss of pdf‐1 induced circadian arrhythmicity under both light–dark (LD) and constant dark (DD) conditions. These defects can be rescued by a genomic copy of the pdf‐1 locus. Our results indicate that PDF‐1 is involved in rhythm generation and in the synchronization to LD cycles, as rhythmic patterns of activity rapidly disappear when pdf‐1 mutants are recorded under both entrained and free‐running conditions. The role of PDF‐2 and the PDF receptors is probably more complex and involves the interaction between the two pdf paralogues found in the nematode.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

Fibroblast growth factor stimulates the gene expression and production of tissue inhibitor of metalloproteinase-1 in bovine granulosa cells

Summary The hormonal control of tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression and production by growth factors, gonadotrophins, and serum factors in cultured bovine granulosa cells (BGC) were investigated. Confluent cultures of BGC were exposed to various factors in a defined medium and levels of TIMP-1 in the conditioned medium were determined by enzyme immunoassay. Basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF) showed potent stimulation of cell proliferation and TIMP-1 production by BGC, while insulin stimulated growth but not TIMP-1 production. Basic FGF stimulated TIMP-1 production and BGC cell proliferation in a dose-dependent manner. A time course of TIMP-1 production showed substantially increased levels between 18 and 24 h in both control and bFGF-stimulated BGC cultures with bFGF-stimulated cultures having markedly higher TIMP-1 production at all time points. Consistent with the TIMP-1 production data, bFGF and aFGF increased the expression of TIMP-1 mRNA as determined by northern blot analysis, while insulin, inhibited TIMP-1 mRNA levels. These results indicate that FGF-induced TIMP-1 production by BGC may support bovine embryo development in vitro.     

An identified dorsal unpaired median neurone and bilaterally projecting neurones exhibiting bovine pancreactic polypeptide-like/FMRFamide-like immunoreactivity in abdominal ganglia of the migratory locust

Summary Three antisera were used to study the distribution and anatomy of bovine pancreatic polypeptide (BPP)-like/FMRFamide-like immunoreactive neurones within the unfused abdominal ganglia of the migratory locust, Locusta migratoria. All the antisera used stained two or more clusters of perikarya, localized anteriorly and posteriorly near the midline within each unfused abdominal ganglion. Double labelling experiments with intracellular dye injection, or differential backfilling, combined with subsequent immunostaining were carried out to identify these neurones. Two of the antisera (antisera 1 and 2, both raised against FMRFamide) stained three groups of midline neurones, located anterior dorsal, anterior ventral and posterior dorsal within the ganglion. Neurones of the former of these two clusters projected via the anterior median nerve to a neurohaemal organ. The posterior cluster of midline cells comprised immunopositive perikarya all but one of which also projected via the anterior median nerve to innervate the neurohaemal organ. Double labelling with Lucifer yellow and antisera 1 and 2 showed that the remaining neurone was the previously identified doral unpaired median (DUM)heart1 neurone. The third antiserum (AK141), also raised against FMRFamide, stained neurones within an anterior dorsal cluster, and in a posterior cluster. Double labelling with differential Co²⁺/Ni²⁺-backfilling and the antiserum 3 (AK141) demonstrated that the large neurones of both clusters belonged to the population of bilaterally projecting neurones (BPNs), including the DUMheart1 neurone. Since the antisera cross-react with BPP and fail to label neurones when preadsorped with BPP or FMRFamide, we conclude that the labelled neurones contain polypeptides of the FMRFamide/BPP-family.     

The dipteran parasitoid Exorista bombycis induces pro‐ and anti‐oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro‐oxidative reactions and activation of anti‐oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione‐S‐transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro‐ and anti‐oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.     

Age‐dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine‐treated mice

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane‐Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2‐treated mice without change in proteins, while it decreased GluN2B protein in P10‐treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5‐treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5‐treated mice. Microdissection of ketamine‐treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I–IV) and an increase in all subunit expressions in deep layers (V–VI) in P5‐ and P10‐treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A‐enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running‐wheel exercise showed age‐ and sex‐dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short‐term NMDAR cortical developmental profile impairments and long‐term motor activity alterations persisting in adulthood. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 315–333, 2015     

Temporal distribution of PBAN‐like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior

A direct enzyme‐linked immunosorbent assay has been developed and applied to the analysis of PBAN immunoreactivity in female hemolymph of the cabbage armyworm, Mamestra brassicae. PBAN‐IR determinations have been carried out with third scotophase insects at different times of the photoperiod. The rhythm of calling and the pattern of pheromone production by third scotophase females at different times of the photoperiod have also been determined. PBAN‐IR and calling are well correlated. However, whereas pheromone titers decrease, both PBAN‐IR levels and percentage of calling females remain high in the last hours of the scotophase. These results are discussed in the context of the regulation of sex pheromone biosynthesis in M. brassicae. Arch. Insect Biochem. Physiol. 40:80–87, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis of the N‐methyl Derivatives of 2‐Aminothiazol‐4(5H)‐one and Their Interactions with 11βHSD1‐Molecular Modeling and in Vitro Studies

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is an enzyme that affects the body's cortisol levels. The inhibition of its activity can be used in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. In this study, we synthesized new derivatives of 2‐(methylamino)thiazol‐4(5H)‐one and tested their activity towards inhibition of 11β‐HSD1 and its isoform – 11β‐HSD2. The results were compared with the previously tested allyl derivatives. We found out that methyl derivatives are weaker inhibitors of 11β‐HSD1 in comparison to their allyl analogs. Due to significant differences in the activity of the compounds, molecular modeling was performed, which was aimed at comparing the interactions between 11β‐HSD1 and ligands differing by substituent at the amine group (allyl vs. methyl). Modeling showed that the absence of the allyl group can lead to the rotation of whole ligand molecule which affects its interaction with the enzyme.     

In vitro and in vivo effects of iron on the expression and activity of glucose 6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase,and glutathione reductase in rat spleen

Iron is an indispensable element for vital activities in almost all living organisms. It is also a cofactor for many proteins, enzymes, and other essential complex biochemical processes. Therefore, iron trafficking is firmly regulated by Hepcidin (Hamp), which is regarded as the marker for iron accumulation. The disruption of iron homeostasis leads to oxidative stress that causes various human diseases, but this mechanism is still unclear. The aim of this study is to provide a better in vivo and in vitro understanding of how long‐term iron overload affects the gene expression and activities of some antioxidant enzymes, such as glucose 6‐phosphate dehydrogenase (G6PD), 6‐phosphogluconate dehydrogenase (6PGD), and glutathione reductase (GR) in the spleen. The findings of this study show that iron overload reduces the gene expression of G6pd, 6pgd, and Gr, but its actual effect was on the protein level.     

White‐tailed deer as the last megafauna dispersing seeds in Neotropical dry forests: the role of fruit and seed traits

Endozoochory is a prominent form of seed dispersal in tropical dry forests. Most extant megafauna that perform such seed dispersal are ungulates, which can also be seed predators. White‐tailed deer (Odocoileus virginianus) is one of the last extant megafauna of Neotropical dry forests, but whether it serves as a legitimate seed disperser is poorly understood. We studied seed dispersal patterns and germination after white‐tailed deer gut passage in a tropical dry forest in southwest Ecuador. Over 23 mo, we recorded ca 2000 seeds of 11 species in 385 fecal samples. Most seeds belonged to four species of Fabaceae: Chloroleucon mangense, Senna mollissima, Piptadenia flava, and Caesalpinia glabrata. Seeds from eight of the 11 species dispersed by white‐tailed deer germinated under controlled conditions. Ingestion did not affect germination of C. mangense and S. mollissima, whereas C. glabrata showed reduced germination. Nevertheless, the removal of fruit pulp resulting from ingestion by white‐tailed deer could have a deinhibition effect on germination due to seed release. Thus, white‐tailed deer play an important role as legitimate seed dispersers of woody species formerly considered autochorous. Our results suggest that more research is needed to fully understand the ecological and evolutionary effects of the remaining extant megafauna on plant regeneration dynamics in the dry Neotropics.     

Temperature‐mediated microhabitat choice and development time based on the pgm locus in the yellow dung fly Scathophaga stercoraria

Temperature can affect the performance of insects through its influence on enzyme function. We report a series of laboratory and field experiments investigating the putatively adaptive temperature‐dependent effects of phosphoglucomutase (pgm) genotype on development time, a central life‐history fitness component, as mediated by the microhabitat choice of ovipositing female yellow dung flies (Scathophaga stercoraria). The allozyme corresponding to the most common pgm allele 3 (approximate 90% natural frequency) had the highest Q ₁₀ and showed higher in vitro activity at warmer temperatures (17–27 °C), whereas the allozyme corresponding to allele 4 (4.5%) showed relatively higher activity at the lowest temperature (7 °C), and the allozyme corresponding to allele 2 (2.5%) showed lower activity throughout. A laboratory experiment revealed that egg development time for allele 3 was shortest (i.e. best) at all temperatures tested, although egg‐to‐adult development time was longest for offspring derived from field females bearing allele 3. Importantly, over 3 years in the field, allele 4 did not increase in frequency late in the season as the temperatures dropped. Although females augmented their proportion of eggs laid on the warmer south slope of a dung pat (adaptive response), the development times of their offspring consistently increased towards the end of the season (maladaptive response). Regardless, females did not systematically bias the pgm composition of eggs laid on the north versus south slopes, as was expected from previous work hypothesizing that females exert microhabitat choice based on the pgm locus. We conclude that, although PGM allozymes differ in activity and the pgm genotype does differentially affect development time, these effects are inconsistent, and not predictable from in vitro allozyme assays, and therefore difficult to reconcile with an adaptive framework of cryptic female choice. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 686–696.     

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half‐smooth tongue sole Cynoglossus semilaevis

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non‐specific esterase (NSE), peroxidase (POD) and mucous‐cell types was evaluated in the gastrointestinal tract of the half‐smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH 2·5) periodic acid Schiff (AB‐PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB‐) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS‐AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.     

Drought and its legacy modulate the post‐fire recovery of soil functionality and microbial community structure in a Mediterranean shrubland

The effects of drought on soil dynamics after fire are poorly known, particularly its long‐term (i.e., years) legacy effects once rainfall returns to normal. Understanding this is particularly important for nutrient‐poor soils in semi‐arid regions affected by fire, in which rainfall is projected to decrease with climate change. Here, we studied the effects of post‐fire drought and its legacy on soil microbial community structure and functionality in a Cistus‐Erica shrubland (Spain). Rainfall total and patterns were experimentally modified to produce an unburned control (natural rainfall) and four burned treatments: control (natural rainfall), historical control (long‐term average rainfall), moderate drought (percentile 8 historical rainfall, 5 months of drought per year), and severe drought (percentile 2, 7 months of drought). Soil nutrients and microbial community composition (ester‐linked fatty acid approach) and functionality (enzyme activities and C mineralization rate) were monitored during the first 4 years after fire under rainfall treatments, plus two additional ones without them (six post‐fire years). We found that the recovery of burned soils was lower under drought. Post‐fire drought increased nitrate in the short term and reduced available phosphorus, exchangeable potassium, soil organic matter, enzyme activities, and carbon mineralization rate. Moreover, drought decreased soil total microbial biomass and fungi, with bacteria becoming relatively more abundant. Two years after discontinuing the drought treatments, the drought legacy was significant for available phosphorus and enzyme activities. Although microbial biomass did not show any drought legacy effect, the proportion of fungi and bacteria (mainly gram‐positive) did, being lower and higher, respectively, in former drought‐treated plots. We show that drought has an important impact on soil processes, and that some of its effects persist for at least 2 years after the drought ended. Therefore, drought and its legacy effects can be important for modeling biogeochemical processes in burned soils under future climate change.