Antennal motor system of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

The organization of the antennal muscles, nerves, and motor neurons has been investigated in the cockroach, Periplaneta americana. Antennal movements have been observed by video analysis, muscle actions have been determined by dissection and direct mechanical testing, and the motor neurons innervating each muscle have been defined with a recently developed selective backfill method. A model of the antennomotor system of Periplaneta has thus been established and compared with that of crickets. Five muscles located within the head capsule insert on the most proximal antennal segment, the scape. By their action, they allow the scape to move in essentially any direction within the dorsoventral and anteroposterior planes. An additional pair of muscles, one dorsal and one ventral, are found within the scape. They insert on the pedicel and move the pedicel in the dorsal-ventral plane. These seven muscles are controlled by at least 17 motor neurons with somata located in the deutocerebrum. By their action, these motor neurons enable cockroaches to move the long flagellum of each antenna through a wide range of positions in the frontal space, medio-laterally, and also allow depression toward the substrate and elevation well above the level of the head. The antennal motor neurons have been classified into five morphological types based on soma and axon location. Each morphological type has been correlated with a particular pattern of muscle innervation and control. The neurites of all motor neurons are located along the medial aspect of the dorsal lobe of the deutocerebrum. This research was supported by grant nos. IBN 96-04629 and 04-22883 from the National Science Foundation.     

Aminergic regulation of pheromone sensillae in the cockroach <Emphasis Type="Italic">Periplaneta americana</Emphasis>

Tremendous adaptability of insects is predominantly provided by fast tuning of physiological functioning of an organism to the permanently changing environmental conditions. One of the mechanisms of plasticity in insects is modulation of performance of their sense organs by neurohormones. Activity of at least three out of four receptor cells located in cockroach pheromonesensitive sensilla is modulated by octopamine. An increase of firing rate of pheromone receptor cells and a decrease of electroantennogram amplitude is accompanied by enhanced behavioral responses of male cockroaches to sex pheromone. The effect of octopamine on reception of a repellent (1,8-cineole, eucalyptol) by an insect is reported for the first time. Simultaneous modulation of responses of receptor cells located in sex specific sensilla to semantically different odorants indicates their cooperation in formation of insect behavior.     

Role of food-derived opioid peptides in regulation of thermosensitivity of <Emphasis Type="Italic">Periplaneta americana</Emphasis> cockroach

The ability of exorphines (relative δ-selective opioid fragments of food-derived peptides) to change the defense response of American cockroaches Periplaneta americana placed in a hot chamber (T= 50°C) was studied. It was found that the ED₅₀ for a fragment of D-ribulose-1,5-bisphosphate carboxylase rubiscolin-5 (YPLDL) doubling the stay time for the insects in the hot chamber was 340 nM/g. The ED₅₀ for wheat gluten exorphine C (YPISL) was 573 nM/g. Cytochropin-4, a fragment of cytochrome B, did not have a significant effect. Comparison of the obtained data with the results of previous experiments with μ-selective exorphines—milk β-casein fragments—led to the conclusion that opioid receptors of various types are equally involved in regulation of cockroach protective behavior. The significance of phytogenous exorphines in formation of the opioid system of insects in the course of evolution is discussed.     

Establishment of an embryonic cell line from the American cockroach <Emphasis Type="Italic">Periplaneta americana</Emphasis> (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process

Despite the pest status and medicinal value of the American cockroach Periplaneta americana, few attempts have been made to establish cell lines from this insect owing to the difficulty of culturing Blattarian cells. Here, we describe the establishment of the RIRI-PA1 line from P. americana embryo tissue following primary culture in modified Grace’s medium containing 20% fetal bovine serum. RIRI-PA1 was found to primarily consist of attached spindle-shaped and giant cells, which attach themselves to their container. The population-doubling time of 40th-passage cells was approximately 84.8 h. The average chromosome number at the 30th passage was 42, with 40% of cells demonstrating substantial variations, with the highest number of variations of 78 and lowest of 24. The identity of RIRI-PA1 was confirmed by comparing the COI gene of these cells to that of P. americana embryo tissue. Telomerase activity decreased in primary cells after 7 d of culture and 5th-passage cells in comparison to embryo tissues; however, compared to the other cultured cells tested, the telomerase activity significantly increased at the 20th passage. We propose that the stagnation periods and cessation of proliferation observed relate to cellular telomerase activity, but the relationship between insect cell proliferation and telomerase as well as the regulatory mechanism involved remains to be elucidated.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Repellency of two terpenoid compounds isolated from <Emphasis Type="Italic">Callicarpa americana</Emphasis> (Lamiaceae) against <Emphasis Type="Italic">Ixodes scapularis</Emphasis> and <Emphasis Type="Italic">Amblyomma americanum</Emphasis> ticks

Callicarpenal (13, 14, 15, 16-tetranor-3-cleroden-12-al) and intermedeol [(4S,5S,7R,10S)-eudesm-11-en-4-ol], isolated from American beautyberry, Callicarpa americana (Lamiaceae), were evaluated in laboratory bioassays for repellent activity against host-seeking nymphs of the blacklegged tick, Ixodes scapularis, and lone star tick, Amblyomma americanum. A strip of organdy cloth treated with test solution was doubly wrapped (treatment on outer layer) around the middle phalanx of a forefinger and ticks released on the fingertip. Callicarpenal and intermedeol, at 155 nmole/cm² cloth repelled 98 and 96% of I. scapularis nymphs, respectively. Dose response tests with I. scapularis nymphs showed no difference in repellency among callicarpenal, intermedeol and Deet (N,N-diethyl-3-methylbenzamide), however, SS220 ((1S,2′S)-2-methylpiperidinyl-3-cyclohexene-1-carboxamide) was significantly more repellent than the other compounds. Callicarpenal, at 155 nmole/cm² cloth, repelled 100 and 53.3% of I. scapularis nymphs at 3 and 4 h, respectively, after the cloth was treated, whereas intermedeol repelled 72.5% of I. scapularis nymphs 3 h after treatment. In comparison with the results obtained with I. scapularis, callicarpenal, intermedeol, Deet and SS220 were less effective against A. americanum. Only intermedeol and SS220 repelled significantly more A. americanum than ethanol controls at 155 nmole compound/cm² cloth. At 1,240 nmole/cm² cloth, callicarpenal and intermedeol repelled 20 and 40% of A. americanum nymphs.     

Effects of pH on NaCl tolerance of American elm (<Emphasis Type="Italic">Ulmus americana</Emphasis>) seedlings inoculated with <Emphasis Type="Italic">Hebeloma crustuliniforme</Emphasis> and <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

In the present study, we investigated the effects of pH treatments on NaCl tolerance in mycorrhizal and non-mycorrhizal American elm. American elm (Ulmus americana) seedlings were inoculated with Hebeloma crustuliniforme, Laccaria bicolor or with both mycorrhizal fungi and subsequently subjected to different pH solutions (pH 3, 6 and 9) containing 0 mM (control) and 60 mM NaCl for 4 weeks. Inoculation with the mycorrhizal fungi did not have a large effect on seedling dry weights when the pH and NaCl treatments were considered independently. However, when the inoculated seedlings were treated with 60 mM NaCl at pH 3 or 6, shoot to root ratios and root hydraulic conductivity were higher compared with non-inoculated plants, likely reflecting changes in seedling water flow properties. At pH 6, transpiration rates were about twofold lower in non-inoculated plants treated with NaCl compared with non-treated controls. For NaCl-treated H. crustuliniforme- and L. bicolor-inoculated plants, the greatest reduction of transpiration rates was at pH 9. Treatment with 60 mM NaCl reduced leaf chlorophyll concentrations more in non-inoculated compared with inoculated plants, with the greatest, twofold, decrease occurring at pH 6. At pH 3, root Na concentrations were higher in inoculated than non-inoculated seedlings; however, there was no effect of inoculation on root Na concentrations at pH 6 and 9. Contrary to the roots, the leaves of inoculated plants had lower Na concentrations at pH 6 and 9, but not at pH 3. The results point to an interaction between ECM fungi and root zone pH for salt tolerance of American elm.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Mariner</Emphasis>-like elements in <Emphasis Type="Italic">Rhynchosciara americana</Emphasis> (Sciaridae) genome: molecular and cytological aspects

Two mariner-like elements, Ramar1 and Ramar2, are described in the genome of Rhynchosciara americana, whose nucleotide consensus sequences were derived from multiple defective copies containing deletions, frame shifts and stop codons. Ramar1 contains several conserved amino acid blocks which were identified, including a specific D,D(34)D signature motif. Ramar2 is a defective mariner-like element, which contains a deletion overlapping in most of the internal region of the transposase ORF while its extremities remain intact. Predicted transposase sequences demonstrated that Ramar1 and Ramar2 phylogenetically present high identity to mariner-like elements of mauritiana subfamily. Southern blot analysis indicated that Ramar1 is widely represented in the genome of Rhynchosciara americana. In situ hybridizations showed Ramar1 localized in several chromosome regions, mainly in pericentromeric heterochromatin and their boundaries, while Ramar2 appeared as a single band in chromosome A.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Co-factors and co-repressors of Engrailed: expression in the central nervous system and cerci of the cockroach, <Emphasis Type="Italic">Periplaneta americana</Emphasis>

In the larval cockroach (Periplaneta americana), knockout of Engrailed (En) in the medial sensory neurons of the cercal sensory system changes their axonal arborization and synaptic specificity. Immunocytochemistry has been used to investigate whether the co-repressor Groucho (Gro; vertebrate homolog: TLE) and the co-factor Extradenticle (Exd; vertebrate homolog: Pbx) are expressed in the cercal system. Gro/TLE is expressed ubiquitously in cell nuclei in the embryo, except for the distal pleuropodia. Gro is expressed in all nuclei of the thoracic and abdominal central nervous system (CNS) of first instar larva, although some neurons express less Gro than others. Cercal sensory neurons express Gro protein, which might therefore act as a co-repressor with En. Exd/Pbx is expressed in the proximal portion of all segmental appendages in the embryo, with the exception of the cerci. In the first instar CNS, Exd protein is expressed in subsets of neurons (including dorsal unpaired medial neurons) in the thoracic ganglia, in the first two abdominal ganglia, and in neuromeres A8–A11 of the terminal ganglion. Exd is absent from the cerci. Because Ultrabithorax/Abdominal-A (Ubx/Abd-A) can substitute for Exd as En co-factors in Drosophila, Ubx/Abd-A immunoreactivity has also been investigated. Ubx/Abd-A immunostaining is present in abdominal segments of the embryo and first instar CNS as far caudal as A7 and faintly in the T3 segment. However, Ubx/Abd-A is absent in the cerci and their neurons. Thus, in contrast to its role in Drosophila segmentation, En does not require the co-factors Exd or Ubx/Abd-A in order to control the synaptic specificity of cockroach sensory neurons.I acknowledge the support of NIH R01 NS45547, NIH-SCORE S06 GM0088224, and RCMI G12 RR03051.     

Micrografting of ASBVd-infected Avocado (<Emphasis Type="Italic">Persea americana</Emphasis>) plants

Shoot tips (meristem plus 2–3 leaf primordia) from in vitro-germinated avocado seedlings of 2 ASBVd-infected cultivars were micrografted in vitro onto decapitated seedlings from 2 ASBVd-free cultivars, and plants were recovered. Shoot tips consisted of two different sizes, i.e., <0.5 mm long and >0.5 mm but <1 mm long. The recovered plants were indexed for ASBVd using RT-PCR. More plants (58.8%) were recovered from scions >0.5 mm than from those that were <0.5 mm (10.3%). RT-PCR demonstrated that ASBVd replicated in all micrografts from infected sources irrespective of the scion size, while no ASBVd was detected in micrografts from plants that tested negative. ASBVd therefore cannot be eliminated by in vitro micrografting.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.