Sociality in <Emphasis Type="Italic">Euglossa</Emphasis> (<Emphasis Type="Italic">Euglossa</Emphasis>) <Emphasis Type="Italic">viridissima</Emphasis> Friese (Hymenoptera,Apidae, Euglossini)

Euglossa viridissima is an orchid bee that forms both solitary and multiple female nests, making it a suitable species for the study of factors leading to diverse degrees of sociality in Euglossines. We conducted observations in eight reused nests (where a first generation of bees had been produced) kept in artificial boxes from the Yucatan Peninsula, Mexico. Five nests were reused (reactivated) by a single female (SFN), two nests reused by a mother and one daughter (MFN1) and one nest reused by the mother and two daughters (MFN2). No single nest was reactivated by unrelated females. The number of foraging trips, their duration and the duration of cell provisioning was not different between SFN and MFN. The overall production of cells per female was not different either between both types of nest. However, in MFN although all females did lay eggs, there was a reproductive skew in favor of the mother (95 and 45% of the brood produced in MFN1 and MFN2 respectively). She showed reproductive control of her daughters through oophagy and displaying threatening behavior when the daughters tried to open a cell where she had laid an egg. Brood losses to parasites (Anthrax sp. (Bom-byliidae) and Hoplostelis bivittata (Megachilidae)) were only found in SFN which possibly reflects and advantage of MFN in this respect. Our results coupled with other studies in Euglossa, reveal that a wide range of social behaviors occur in this genus, from solitary and communal to primitive reproductive division of labor. Multiple factors involving different levels of pressure imposed by food availability and parasites may favor such a diverse range of nesting behaviors. Interestingly, female associations in E. viridissima seem a result of kin selection that is enforced by coercion from mother females on their daughters. More studies are needed to shed light upon the social organization of Euglossa and other Euglossines and on their phylogenetic relationships in order to trace the origins of eusociality in Apidae. Received 12 February 2008; revised 25 June 2008; accepted 17 July 2008.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> assays for osteoclast apoptosis

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

A new species of <Emphasis Type="Italic">Macroptilium</Emphasis> (Benth.) Urb. (<Emphasis Type="Italic">Leguminosae</Emphasis>: <Emphasis Type="Italic">Papilionoideae</Emphasis>: <Emphasis Type="Italic">Phaseolinae</Emphasis>) from North-Eastern Brazil

Summary  A new species of Macroptilium sect. Microcochle (Benth.) J. A. Lackey is herein described from the states of Piauí and Bahia, Brazil. Macroptilium cochleatum is characterized by few-flowered inflorescences, calyx teeth longer than the tube, a tightly four-coiled keel, and linear, patent fruits. The discovery of this unique Macroptilium species, with its distally coiled keel, expands the diagnostic features of the genus. A key to the Brazilian species of sect. Microcochle is provided.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Interspecific hybridization of <Emphasis Type="Italic">Cucumis anguria</Emphasis> and <Emphasis Type="Italic">C. zeyheri via</Emphasis> embryo-rescue

Embryo-rescue was used to facilitate interspecific hybridization of Cucumis anguria L. and C. zeyheri Sond. Embryos were excised from developing fruits at one week intervals for six weeks after hand pollination. Medium containing coconut water was the most suitable for initial germination, and a medium with ascorbic acid was the best for embryo development and plant recovery. Viable plants were obtained from embryos and these plants showed morphological characteristics different from both parents. The analysis of the leucine aminopeptidase (LAP) locus revealed three hybrid types, H1.1, H1.2 and H2.     

Assessment of genetic stability of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Dictyospermum ovalifolium</Emphasis>

In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of 2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from 200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92 %) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups. Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred fifty plants were successfully established.     

Genetic relationships among <Emphasis Type="Italic">Hystrix patula,H. duthiei</Emphasis> and <Emphasis Type="Italic">H. longearistata</Emphasis> according to meiotic studies and genome-specific RAPD assay

Hybrids including Hystrix patula, H. duthiei and H. longearistata were obtained and genetic relationships among them were studied. Meiotic pairing in hybrids of H. duthiei × Psathyrostachys juncea (Ns), H. longearistata × Psa. juncea (Ns), Leymus multicaulis (NsXm) × H. duthiei, L. multicaulis (NsXm) × H. longearistata, Elymus sibiricus (StH) × H. patula, Roegneria ciliaris (StY) × H. patula, R. ciliaris (StY) × H. duthiei and R. ciliaris (StY) × H. longearistata averaged 5.76, 5.44, 11.94, 10.88, 10.08, 3.57, 0.46 and 0.90 bivalents per cell, respectively. The results indicated that H. duthiei and H. longearistata had the NsXm genomes of Leymus, while H. patula contained the StH genomes and had a low genome affinity with the StY genomes of Roegneria. Results of genome-specific RAPD assay were comparable with the chromosome pairing data. According to the genomic system of classification in Triticeae, H. patula should be considered as Elymus hystrix L., while H. duthiei and H. longearistata as Leymus duthiei and Leymus duthiei ssp. longearistata, respectively.     

Ca<Superscript>2+</Superscript> reduces the effect of hypoxia in mosses <Emphasis Type="Italic">Mnium undulatum</Emphasis> and <Emphasis Type="Italic">Polytrichum commune</Emphasis>

Gametophores of mosses Mnium undulatum and Polytrichum commune were submerged in distilled water or in calcium chloride solution (0.9 mM Ca²⁺) to induce hypoxia. The net photosynthetic (P_N) and dark respiration rate (R_D) were measured in the air containing 300–400 μmol(CO₂)·mol⁻¹(air) and 0.21 mol(O₂)·mol⁻¹(air). P_N of M. undulatum gametophores decreased to 58 % of the control after 1-h submersion in water, whereas to 80 % of the control in P. commune gametophores. A smaller decrease in P_N was observed when the gametophores were immersed in CaCl₂ solution. In hypoxia, R_D in the tested mosses species was a little higher than in the control.     

Methods designed for the identification and characterization of<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> chromatin assembly mutants in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks. Published: July 3, 2003     

A new tip homolog, <Emphasis Type="Italic">ShTIP</Emphasis>, from <Emphasis Type="Italic">Salicornia</Emphasis> shows a different involvement in salt stress compared to that of TIP from <Emphasis Type="Italic">Arabidopsis</Emphasis>

To obtain an insight into the comprehensive molecular characteristics of the salt tolerance mechanism, we performed a screening for salt inducible genes in a halophytic plant, Salicornia herbacea, using mRNA differential display. A comparative analysis of gene expression in Salicornia grown in control and salt-stressed conditions led to the detection of a gene that was induced by salt. Both sequence analysis and a subsequent database search revealed that this gene was highly homologous to tonoplast intrinsic proteins (TIPs) from a variety of plant species. This gene, designated as ShTIP, is 1014 bp in size and contains a coding region of 762 nucleotides, which encodes a protein of 254 amino acids. Northern blot analysis revealed that ShTIP was predominantly expressed in shoots under normal conditions. However, salt stress induced high expression of ShTIP in both the shoots and roots. The expression of ShTIP in a salt-sensitive calcineurin-deficient yeast mutant (cnbΔ) resulted in a resistance to the high salt conditions. In addition, we compared the expression of a TIP gene in Arabidopsis with that of ShTIP under different conditions and found that the Salicornia TIP has a different regulatory mechanism for adapting to salt stress conditions compared with the glycophyte Arabidopsis TIP. These results indicate that ShTIP plays an important role in salt tolerance.     

Photosynthetic characteristics of an endangered species <Emphasis Type="Italic">Camellia nitidissima</Emphasis> and its widespread congener <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Saturation (SI) and compensation (CI) irradiances [μmol(photon) m⁻² s⁻¹] were 383.00±18.40 and 12.95±0.42 for wild C. nitidissima (in mid-July) and 691.00±47.39 and 21.91±1.28 for wild C. sinensis, respectively. C. nitidissima is a shade tolerant species, whereas C. sinensis has a wide ecological range of adaptability to irradiance. Both wild and cultivated C. nitidissima demonstrated low maximum net photosynthetic rate, maximum carboxylation rate, maximum electron transfer rate, and SI, which indicated low photosynthesis ability of leaves that were unable to adapt to strong irradiance environment. Both C. nitidissima and C. sinensis demonstrated strong photosynthetic adaptabilty in new environments. Hence proper shading may raise photosynthetic efficiency of cultivated C. nitidissima and promote its growth.     

Population- and sociogenetic structure of the leaf-cutter ant <Emphasis Type="Italic">Atta colombica</Emphasis> (Formicidae,Myrmicinae)

Relatedness and genetic variability in colonies of social insects are strongly influenced by the number of queens present and the number of matings per queen, but also by the genetic variability in the population. Thus, multiple paternity will enhance within-colony genetic variability more strongly when the males a queen mates with are unrelated. To study the kin-structure within colonies of the leaf-cutter ant Atta colombica and the population structure of this species around Barro Colorado Island, Panama, we developed five polymorphic microsatellite loci with a range of three to 17 alleles in At. colombica, all of which cross-amplify in other higher attines as well. The average effective mating frequency calculated from four-locus microsatellite genotypes was 1.89 ± 0.12 (harmonic mean ± SE) and thus slightly lower than the average observed mating frequency of 2.50 ± 0.11 (arithmetic mean ± SE) over the 55 colonies studied, confirming former studies that utilized fewer loci. The discrepancy between observed mating frequency and effective mating frequency is most probably due to paternity skew within colonies. The study population proved to be genetically diverse and in Hardy-Weinberg equilibrium, suggesting random mating within the study area. No population substructure was observed, neither considering nuclear (global F _ST = 0.011 ± 0.003 SE) nor mitochondrial markers (mean Φ_ST = 0.008). Consequently, gene flow is obviously promoted by both sexes across the range investigated here. Thus, multiple mating and long-distance dispersal appear to be two interconnected behavioural mechanisms to create and maintain genetic diversity in At. colombica. The advantages of this system are partly offset by paternity skew and the non-zero relatedness among colony fathers found in the study population. Received 18 March 2008; revised 14 July 2008; accepted 18 July 2008.     

Expression pattern of <Emphasis Type="Italic">serine protease inhibitor kazal type 3 (Spink3)</Emphasis> during mouse embryonic development

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3 ( +/lacZ ) knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3 ( lacZ ) expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3 ( lacZ ) expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3 ( lacZ ) activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.     

<Emphasis Type="Italic">In vitro</Emphasis> direct organogenesis and regeneration of <Emphasis Type="Italic">Medicago sativa</Emphasis>

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm⁻³ thidiazuron (TDZ) and 3 mg dm⁻³ AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm⁻³ α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.     

<Emphasis Type="Italic">Psithyrus</Emphasis> females do possess wax glands

In behavioural experiments and histological investigations we could demonstrate that the previously widespread generalization that females of the social parasite genus Bombus subgenus Psithyrus do not possess wax glands is not justified. Wax pieces that accumulated on the abdomina of Bombus vestalis, B. sylvestris, and B. bohemicus females which were prevented from self-grooming provided the first evidence for wax production in these species. A histological analysis of epithelial tissues confirmed the existence of epithelial wax glands. Received 17 September 2007; revised 12 March and 2 July 2008; accepted 4 July 2008.     

Establishment of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for <Emphasis Type="Italic">Fortunella crassifolia</Emphasis>

Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm⁻³ kanamycin and 300 mg dm⁻³ cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm⁻³ kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved.     

Environmental factors influencing daily foraging activity of <Emphasis Type="Italic">Vespula germanica</Emphasis> (Hymenoptera,Vespidae) in Mediterranean Australia

In social insects, workers forego reproduction in favour of foraging and other tasks to promote growth of the whole colony. Maximising individual work effort is limited by the physical constraints on foraging outside the nest. Previous studies of factors influencing activity in social insects suggest that light intensity, ground and air temperature, relative humidity, solar radiation, wind speed and/or rainfall may be important. This study aimed to determine which environmental factors influence foraging in an invasive social insect, Vespula germanica, which has been introduced to Australia, where it experiences a hotter and drier climate than in its native range. Activity was measured in terms of foraging traffic, with nests from a range of locations being monitored. Results indicate that the onset of rain reduces activity by approximately 30%, but foraging returns to previous levels immediately after rain stops. Foraging time is correlated with duration of daylight, with wasp daily activity being on average 22 min longer than the time between sunrise and sunset. Low light was found to restrict wasp activity, as were low and high temperatures. A linear mixed-effects model developed to explain the influence of these variables on numbers of foragers was highly significant. Under hot conditions, V. germanica individuals thermoregulate their body temperature by regurgitating water. They also use water in evaporative cooling to keep nests at optimum temperatures. Thus, in this species, hot temperatures increase the need for water, and so populations may be severely impacted in seasonally hot regions where water is limited. Received 30 March 2007; revised 10 March 2008; accepted 9 April 2008.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.