Gene silencing in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: dsRNA and siRNA parental silencing of the <Emphasis Type="Italic">Distal-less</Emphasis> gene

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.     

In silico cloning of genes encoding neuropeptides, neurohormones and their putative G-protein coupled receptors in a spider mite

The genome of the spider mite was prospected for the presence of genes coding neuropeptides, neurohormones and their putative G-protein coupled receptors. Fifty one candidate genes were found to encode neuropeptides or neurohormones. These include all known insect neuropeptides and neurohormones, with the exception of sulfakinin, corazonin, neuroparsin and PTTH. True orthologs of adipokinetic hormone (AKH) were neither found, but there are three genes encoding peptides similar in structure to both AKH and the AKH-corazonin-related peptide. We were also unable to identify the precursors for pigment dispersing factor (PDF) or the recently discovered trissin. However, the spider mite probably does have such genes, as we found their putative receptors. A novel arthropod neuropeptide gene was identified that shows similarity to previously described molluscan neuropeptide genes and was called EFLamide. A total of 65 putative neuropeptide GPCR genes were also identified, of these 58 belong to the A-family and 7 to the B-family. Phylogenetic analysis showed that 50 of them are closely related to insect GPCRs, which allowed the identification of their putative ligand in 39 cases with varying degrees of certainty. Other spider mite GPCRs however have no identifiable orthologs in the genomes of the four holometabolous insect species best analyzed. Whereas some of the latter have orthologs in hemimetabolous insect species, crustaceans or ticks, for others such arthropod homologs are currently unknown.     

Effects of growth hormone on the ultrastructure of bovine preimplantation embryos

Growth hormone (GH) has recently been shown to promote the development of preimplantation embryos. The aim of our study was therefore to analyze the effects of GH on the morphology and ultrastructure of the cells of bovine preimplantation embryos produced by in vitro fertilization (IVF). In order to determine the physiologically optimal morphology of blastocysts, ex vivo embryos obtained by uterine flushing were also included in the study. As shown by transmission electron microscopy, treatment with GH induced the elimination of glycogen storage in cells of the inner cell mass of 7-day-old embryos. GH also stimulated the exocytosis of lipid vesicles in the inner cell mass and trophectoderm cells of these embryos. Quantitative analysis of micrographs demonstrated a higher volume density of embryonic mitochondria in 7-day-old embryos cultured with GH than in control embryos. Treatment with GH regularly resulted in an improvement of the ultrastructural features of embryos produced in vitro, thus resembling the morphology of ex vivo embryos. Scanning electron-microscopy studies demonstrated that GH altered the structure and the pore size of the zona pellucida of blastocysts. Our studies imply that GH can modulate carbohydrate, lipid, and energy metabolism and influence transportation processes in the early IVF embryo.This work was supported by the Deutsche Forschungsgemeinschaft (FOR 478/1)     

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.     

Effects of prostaglandin PGE2 on the synthesis of placental proteins and human placental lactogen (HPL)

The biosynthesis of placental proteins and placental lactogen (HPL) was studied $\text{in vitro}$ in 10–12 week, 16–18 week and term human placenta in the presence and absence of PGE_2α. The highest ¹⁴C-leucine incorporation was detected in 10 to 12 weeks old placentas. Addition of PGE_2α to the induction medium depressed the rate of incorporation of ¹⁴C-leucine into placental proteins on a dose dependent manner. Placentas most sensitive to this action of PGE_2α were those obtained at 18 weeks gestation followed by placentas at term. $\text{In vivo}$ application of PGE_2α for tharapeutic induction of abortions resulted in the marked inhibition of placental protein synthesis $\text{in vitro}$.     

Effects of zinc on the mineralization of bone nodules from human osteoblast-like cells

Zinc is an important mineral that is required for normal bone development. However, the direct effects of zinc on the mineralization of bone cells of human origin are not clear. The objective of this study was to determine the effects of zinc on the differentiation of SaOS-2 human osteoblast-like cells and the formation of mineralized bone nodules. Cells were cultured for 8 d and then transferred to zinc-free medium and treated with varying concentrations (0–50 μM) of zinc. Alkaline phosphatase (ALP) activity was used as a measure of osteoblast differentiation, and bone nodules were detected by von Kossa staining. After 4, 6, and 8 d of treatment, zinc increased ALP activity at 1 and 10 μM, but decreased activity at 50 μM. After 9 d of treatment, zinc increased both the number and area of mineralized bone nodules at low concentrations (1 and 10 μM), but decreased both at higher concentrations (25 and 50 μM). These findings demonstrate that zinc has biphasic effects on the differentiation and mineralization of human osteoblast-like cells.     

Macrophage colony stimulating factor (CSF-1) is a central growth factor of goldfish macrophages

We recently characterized macrophage colony stimulating factor (CSF-1) of fish (the goldfish). Here, we report for the first time that goldfish CSF-1 acts through the CSF-1 receptor by showing loss of CSF-1 function in CSF-1R knockdown monocytes using RNAi, and demonstrate that goldfish CSF-1 administration in vivo increases the amount of circulating monocytes in blood. We also show that conditioned supernatants from goldfish fibroblast cultures induced the proliferation of goldfish monocytes indicating that, like in mammals, teleost fibroblasts are an important producer of CSF-1. The continuous addition of recombinant CSF-1 to primary goldfish macrophage cultures stabilized and extended their longevity and resulted in a long-term culture of functional macrophages capable of mounting a potent nitric oxide response upon activation with goldfish recombinant TNF-alpha.     

Efficient transgenesis in farm animals by lentiviral vectors

Microinjection of DNA is now the most widespread method for generating transgenic animals, but transgenesis rates achieved this way in higher mammals are extremely low. To address this longstanding problem, we used lentiviral vectors carrying a ubiquitously active promoter (phosphoglycerate kinase, LV-PGK) to deliver transgenes to porcine embryos. Of the 46 piglets born, 32 (70%) carried the transgene DNA and 30 (94%) of these pigs expressed the transgene (green fluorescent protein, GFP). Direct fluorescence imaging and immunohistochemistry showed that GFP was expressed in all tissues of LV-PGK transgenic pigs, including germ cells. Importantly, the transgene was transmitted through the germ-line. Tissue-specific transgene expression was achieved by infecting porcine embryos with lentiviral vectors containing the human keratin K14 promoter (LV-K14). LV-K14 transgenic animals expressed GFP specifically in basal keratinocytes of the skin. Finally, infection of bovine oocytes after and before in vitro fertilization with LV-PGK resulted in transgene expression in 45% and 92% of the infected embryos, respectively.