The bulbus is a part of the unique functional penis (endophallus) in the genus Apis and was studied in Apis mellifera drones. The bulbus consists of a thin cuticular membrane, covered by four different epithelia (dorsal epithelium, anterior epithelium, ventral epithelium I and ventral epithelium II). One day before eclosion, pupae have no sclerotised plates in the bulbus lumen. In emerging drones, however, the lumen contains tanned plate-like structures, the chitin plates, subjacent are small droplets. The chitin plates are connected to each other by a transparent matrix. They consist of globular sclerotized structures which are connected by a network of fibrils. In young adult drones, the dorsal and lateral epithelia consist of highly columnar gland cells which decrease in thickness from the age of 6–12 days from 140 to 20 μm. The ultrastructural analysis combined with histochemistry shows that the gland cells secrete proteinaceous droplets towards the lumen which pass through the cuticle and the pores of the chitin plates and accumulate in the matrix. In sexually mature drones, all secretions have merged to a rigid connective substance, which extends laterally as a thin tapering membrane. The ventral epithelia I and II have no secretory function. The ventral epithelium II has a mucus-like content and several invaginations towards the lumen of the bulbus which enables widening of the bulbus orifice to discharge the bulbus secretions filled with viscous mucus via the cervix into the queen during the mating process.

We tested whether flow cytometry can be used for assessment of viability of honey bee (Apis mellifera) sperm. The method was used to detect possible competition between the sperm of different drones. The flow cytometry analysis of semen stained with SYBR-14/propidium iodide revealed significant differences between fresh and freeze-thawed samples. The identification of populations corresponding to viable and nonviable sperm allowed us to assess the sperm viability. The comparison of single-drone semen with mixed semen of two unrelated drones showed that sperm viability was not affected by mixing, but there were differences between mixed and unmixed semen in side scatter, which correlates with shape and optical homogeneity of particles. The proportion of particles in different populations also was affected by mixing of the semen. The results suggest that there are interactions between ejaculates of different drones, possibly related to sperm competition.

Honeybees are the most important pollinators of agricultural and horticultural crops. Most fruit, small seed and many vegetable crops require pollination for the production of economic yields. The value of the honeybee as a pollinator is far greater than its value as a honey producer. Not all crops need pollination. Some can produce fruit without fertilization of the flower. Some flowers are self pollinated, which means that pollen is transferred from the anther to the stigma of the same flower or flowers on the same plant variety. Although this transfer can be achieved by wind or rain, insect pollinators are the most effective. Of all the insects, hive bees are the most practical for crop pollination can be reared in sufficient numbers and placed in orchards wherever and whenever required for effective pollination. It has been found that the use of hive bees results in a manifold increase in yields and an improvement in the quality of produce.

Complementary sex determination in Hymenoptera implies that heterozygosity at the sex locus leads to the development of diploid females, whereas hemizygosity results in haploid males. Diploid males can arise through inbreeding. In social species, these pose a double burden on colony fitness, from significant reduction in its worker force and through being less viable and fertile than haploid males. Apart from being “misfits”, diploid males are of interest to assess molecular correlates for possibly ploidy-related bionomic differences. Herein, we generated suppression subtractive cDNA libraries from newly emerged haploid and diploid males of the stingless bee Melipona quadrifasciata to enrich for differentially expressed genes. Gene Ontology classification revealed that in haploid males more DEGs were related to stress responsiveness, biosynthetic processes, reproductive processes and spermatogenesis, whereas in diploid ones differentially expressed genes were associated with cellular organization, nervous system development and amino acid transport were prevalent. Furthermore, both libraries contained over 40 % ESTs representing possibly novel transcripts. Quantitative RT-PCR analyses confirmed the differential expression of a representative DEG set in newly emerged males. Several muscle formation and energy metabolism-related genes were under-expressed in diploid males. On including 5-day-old males in the analysis, changes in transcript abundance during sexual maturation were revealed.

Summary

In recent years there has been growing appreciation of the unexpectedly large genetic diversity of microbes in the environment. This diversity has important implications for our understanding of photosynthesis in populations and in the environment. Conventional methodologies often cannot effectively capture this aspect. This chapter describes new approaches including comparative genomic and metagenomic approaches combined with a more detailed understanding of the metabolism and functionalities of cyanobacteria. This approach, which can be defined broadly as “functional ecogenomics” is partly motivated by the availability of high-throughput sequence data, which are steadily being acquired. The focus is on unicellular cyanobacteria in the hot spring microbial mats of Yellowstone National Park, which are primary producers in this prokaryotic community. We took a three-pronged approach, in which we (a) acquired complete genome sequences from two dominant Synechococcus sp. and carried out a comparative genomic analysis to understand the functional differences between these temperature adapted isolates; (b) produced a metagenome dataset that allows us to place genomic information in the context of the community within which these cyanobacteria grow and evolve; and (c) obtained pure isolates of some dominant organisms that allows us to manipulate them in a well-defined laboratory setting. In situ transcriptomics also allowed quantification of transcripts during the diel cycle. These diverse approaches and the ability to measure environmental parameters such as light and O₂ levels allows us detailed insight into the microbial mat system. Such an approach could be used to study a wide array of photosynthetic microorganisms as populations and interacting communities. As sequencing capacity, single cell capture techniques, proteomics and imaging techniques become more widely accessible we expect to obtain ever more detailed information about natural communities.

Males, queens and workers of stingless bees show differences in external morphology, behaviour and roles within a colony. In addition, each individual has a cuticular chemical signature responsible for mutual communication that is essential for maintaining the integrity of the colony. In this paper we characterize the cuticular hydrocarbon composition of newly emerged diploid and haploid males, workers and virgin queens of Melipona quadrifasciata by gas chromatography-mass spectrometry (GC/MS) analysis. This is the first time that the cuticular profile of diploid males in a species of stingless bee has been characterized. We found differences in the cuticular hydrocarbon composition among males, workers and virgin queens, recording both qualitative and quantitative differences among individuals of different phenotypes. However, no compound was found exclusively in diploid males. The cuticular chemical profiles of haploid and diploid males were very similar to those of workers. Moreover, the cuticular lipids of males and workers were significantly different from those of queens. Tricosane, pentacosene-2 and 7-methyl-heptacosane were the compounds responsible for this significant separation. This result correlates with the behavioural and morphological differences among these phenotypes.

Production of hybrids to obtain higher yields is newer breeding method. To produce hybrid seed, the flowers on the male-sterile lines must be visited by nectar seeking pollen coated insects that have earlier visited the flowers of male-fertile plants. Now-a-days there is an increasing demand for hybrid seed of various crops. For hybrid seed production, parental cultivars are selected and crossed to produce off-spring of increased size, uniform volume, quality in earliness or resistance to unfavourable environmental factors when grown commercially. The production of these hybrid seed crops needs special techniques to prevent self-pollination and encourage cross-pollination favourable by insects.

Comparative analyses of the amino acid sequence of the N-terminus of methacrylate reductase (50 kDa) from the anaerobic bacterium Geobacter sulfurreducens AM-1 revealed significant similarity to the sites of flavocytochromes c and flavin-containing proteins of anaerobic bacteria from the genera Anaero-myxobacter, Campylobacter, Desulfatibacillum, Desulfuromonas, Geobacter, Parasutterella, Shewanella, Sulfurospirillum, Sutterella, and Wolinella (belonging to four classes of the Proteobacteria), Denitrovibrio of the phylum Deferribacteres, Desulfitobacterium of the phylum Firmicutes, Eggerthella and Slackia of the phylum Actinobacteria, and Spirochaeta of the phylum Spirochaetes. High homology was also revealed between the methacrylate reductase amino acid sequence and the flavoproteins of aerobic bacteria Frankia and Gordonia from the phylum Actinobacteria and of the archaeon Haloterrigena turkmenica from the phylum Euryarchaeota. Among 11 Geobacter strains with known genomes, only G. lovleyi SZ capable of chlororespiration was found to contain an amino acid fragment of flavocytochrome c with pronounced similarity (80%) to the N-terminus of methacrylate reductase. The physiological properties common to these bacteria are discussed. Molecular masses and the hypothetical functions and localization of the homologous proteins are analyzed. The grouping of proteins according to the phylogenetic affiliation of their owners is discussed. Hypotheses concerning the distribution and evolutionary role of such proteins in microorganisms are suggested.

Key Points

Genomic sequencing from a single cell became feasible with the introduction of the multiple displacement amplification (MDA) method, which generates micrograms of amplified DNA.

Methods have improved over the past 10 years to isolate single cells, amplify the DNA by MDA for use in sequencing, and assemble genomes from those single cells.

A vast range of novel microorganisms will now be amenable to genomic sequencing directly from single cells, eliminating the need to develop culture methods in order to obtain sufficient DNA template.

Single-cell sequencing is increasingly being used in combination with metagenomic sequencing to assemble individual genomes and analyse complex microbial communities.

The hydrothermal vent mussel Bathymodiolus puteoserpentis (Mytilidae) from the Mid-Atlantic Ridge hosts symbiotic sulfur- and methane-oxidizing bacteria in its gills. In this study, we investigated the activity and distribution of these two symbionts in juvenile mussels from the Logatchev hydrothermal vent field (14°45′N Mid-Atlantic Ridge). Expression patterns of two key genes for chemosynthesis were examined: pmoA (encoding subunit A of the particulate methane monooxygenase) as an indicator for methanotrophy, and aprA (encoding the subunit A of the dissimilatory adenosine-5′-phosphosulfate reductase) as an indicator for thiotrophy. Using simultaneous fluorescence in situ hybridization (FISH) of rRNA and mRNA we observed highest mRNA FISH signals toward the ciliated epithelium where seawater enters the gills. The levels of mRNA expression differed between individual specimens collected in a single grab from the same sampling site, whereas no obvious differences in symbiont abundance or distribution were observed. We propose that the symbionts respond to the steep temporal and spatial gradients in methane, reduced sulfur compounds and oxygen by modifying gene transcription, whereas changes in symbiont abundance and distribution take much longer than regulation of mRNA expression and may only occur in response to long-term changes in vent fluid geochemistry.