Microsatellite markers in the primitive termite Mastotermes darwiniensis

We developed 10 microsatellite loci in the primitive termite Mastotermes darwiniensis. The number of alleles per locus ranged from four to 15, and the expected heterozygosites spanned from 0.21 to 0.90, in a sample of 40 workers collected from the Northern Territory, Australia. We also determined that only two loci amplified in five other termite species. The low frequency of cross‐amplification probably resulted from the high level of phylogenetic divergence between M. darwiniensis and the other taxa. Thus, although the loci are not widely applicable, they should prove effective in elucidating the genetic structure of M. darwiniensis populations.     

The spermatozoon of arthropoda. XXX. The multiflagellate spermatozoon in the termite Mastotermes darwiniensis

In this paper the spermatozoon of the termite Mastotermes darwiniensis is described. It is the first example of a multiflagellate sperm cell in animals. The sperm consists of a conical head and 100 flagella. Other remarkable features of this sperm cell are the absence of an acrosome, the presence of centrioles containing doublet microtubules instead of triplets, and the presence of axonemes devoid of central tubules and with doublets bearing only one arm. The flagella are feebly motile.     

Population and colony genetic structure of the primitive termite Mastotermes darwiniensis

The termite Mastotermes darwiniensis is the sole extant member of its family and occupies the basal position in the phylogeny of the eusocial order Isoptera. In this study, we investigated the micro- and macrogeographic genetic structure of M. darwiniensis in its native range in Australia. A total of 1591 workers were sampled from 136 infested trees in 24 locales. Each locale was separated by 2-350 km, and these locales were found within two broader geographic regions approximately 1500 km apart. The multilocus genotypes of all termites were assayed at six polymorphic microsatellite loci. The genetic data indicated that colonies typically fed on multiple trees within locales and extended over linear distances of up to 320 m. Single colonies were frequently headed by multiple reproductives. Workers were highly related (r = 0.40) and substantially inbred (f = 0.10). Thus, M. darwiniensis colonies are characterized by the input of alleles from multiple reproductives, which sometimes engage in consanguineous matings. Our analyses of population genetic structure above the level of the colony indicated that locales and regions were significantly differentiated (theta(locale) = 0.50, theta(region) = 0.37). Moreover, locales showed a pattern of genetic isolation by distance within regions. Thus, M. darwiniensis populations display restricted gene flow over moderate geographic distances. We suggest that the genetic patterns displayed by M. darwiniensis result primarily from selective pressures acting to maintain high relatedness among colonymates while allowing colonies to grow rapidly and dominate local habitats.     

Symbiosestudien III: Die intrazellluare bakterien- symbiose von mastotermes darwiniensis froggatt (isoptera).

Ohne Zusammenfassung     

Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis

Animals are often exposed to or can ingest heavy metals along with their food. Therefore, we tested whether the hindgut microbiota of Mastotermes darwiniensis possesses the capability to form methyl mercury. The termite M. darwiniensis (Isoptera) was fed with saw dust containing different concentrations of inorganic mercury. Methyl mercury was determined by purge‐and‐trap capillary gas chromatography‐atom fluorescence spectrometry (CGC‐AFS) using ethyl mercury chloride as the internal standard. Total mercury concentrations were determined in the termite tissue by inductive coupled plasma‐isotope dilution mass spectrometry (ICP‐IDMS) after microwave‐assisted digestion. The obtained results showed in vivo methyl mercury production in terrestrial insects for the first time. Desulfovibrio intestinalis isolated from M. darwiniensis was identified as a biomethylating species of the intestinal microbiota.     

Ectobiotic spirochetes of flagellates from the termite Mastotermes darwiniensis: attachment and cyst formation

The association of the gut flagellates Mixotricha paradoxa and Deltotrichonympha sp. from the termite Mastotermes darwiniensis with ectobiotic spirochetes and bacterial rods is investigated with light and electron microscopy. Treatment with different chemicals disturbing molecular interactions and use of the freeze-fracture and freeze-etch technique show that hydrophobic interactions and integral membrane proteins seem to be involved in the firm attachment at the contact sites. Application of antibiotics reduces the number of ectobionts and leads to a disintegration of the cortical attachment systems. As a result Mixotricha becomes spherical and immotile. In both flagellates the antibiotics have a further effect: they lead to a transformation of some of the spirochetes into cystic bodies. Cyst formation of ectobiotic spirochetes is here reported for the first time. Starvation has a similar but less dramatic influence than antibiotics. The cysts contain protoplasmic cylinders in the periphery and sometimes larger central bodies. Production of dormant cystic forms may be a survival mechanism under hostile conditions.     

Genome shrinkage and loss of nutrient-providing potential in the obligate symbiont of the primitive termite Mastotermes darwiniensis

Beneficial microbial associations with insects are common and are classified as either one or a few intracellular species that are vertically transmitted and reside intracellularly within specialized organs or as microbial assemblages in the gut. Cockroaches and termites maintain at least one if not both beneficial associations. Blattabacterium is a flavobacterial endosymbiont of nearly all cockroaches and the termite Mastotermes darwiniensis and can use nitrogenous wastes in essential amino acid and vitamin biosynthesis. Key changes during the evolutionary divergence of termites from cockroaches are loss of Blattabacterium, diet shift to wood, acquisition of a specialized hindgut microbiota, and establishment of advanced social behavior. Termite gut microbes collaborate to fix nitrogen, degrade lignocellulose, and produce nutrients, and the absence of Blattabacterium in nearly all termites suggests that its nutrient-provisioning role has been replaced by gut microbes. M. darwiniensis is a basal, extant termite that solely retains Blattabacterium, which would show evidence of relaxed selection if it is being supplanted by the gut microbiome. This termite-associated Blattabacterium genome is ～8% smaller than cockroach-associated Blattabacterium genomes and lacks genes underlying vitamin and essential amino acid biosynthesis. Furthermore, the M. darwiniensis gut microbiome membership is more consistent between individuals and includes specialized termite gut-associated bacteria, unlike the more variable membership of cockroach gut microbiomes. The M. darwiniensis Blattabacterium genome may reflect relaxed selection for some of its encoded functions, and the loss of this endosymbiont in all remaining termite genera may result from its replacement by a functionally complementary gut microbiota.     

Isolation of Desulfovibrio intestinalis sp. nov. from the hindgut' of the lower termite Mastotermes darwiniensis.

A Gram-negative, anaerobic sulfate-reducing bacterium was isolated from hindgut contents of the lower termite Mastotermes darwiniensis Froggatt (strain KMS2). Strain KMS2 is motile by a single polar flagellum. The isolate possesses desulfoviridin and catalase activity. The G+C content of its DNA is in the range of 54.5-55.5 mol% (strain KMS2). It respires hydrogen and different low molecular weight organic compounds in the presence of sulfate, thiosulfate, and sulfite, and also oxygen. The isolated strain ferments pyruvate. Fastest growth with a doubling time of 12.5 h was obtained at 37 degrees C and not at 28 degrees C, the temperature at which the termites were grown. The isolate showed a 16S rDNA sequence homology of 95.9% to Desulfovibrio desulfuricans ATCC 27774 and a DNA-DNA homology of 44.6% to D. desulfuricans Essex 6 (type strain). Based on its biochemical properties and 16S rDNA sequence, the isolate was assigned to a new species named Desulfovibrio intestinalis.     

Association between caste and genotype in the termite Mastotermes darwiniensis Froggatt (Isoptera: Mastotermitidae)

Abstract Termite workers and soldiers differ markedly in their morphology and behaviour. We sought evidence for genetic influences on caste determination in the giant northern termite, Mastotermes darwiniensis , by investigating if workers and soldiers from the same colony differed genetically. The genotypes of 795 termites from 11 distinct colonies were assayed at six polymorphic microsatellite loci. We found that the multilocus genotypes of workers and soldiers from 8 of the 11 colonies did not differ significantly. Thus, the majority of the data provided no evidence for a genetic association with caste in workers and soldiers of M. darwiniensis . However, the genotype frequencies of workers and soldiers from three colonies differed, suggesting that genotype is occasionally associated with caste in this species. The genetic differentiation of castes within these colonies could reflect differences in the propensities of termites with distinct genotypes to develop into particular castes and provide a selective advantage to colonies headed by multiple reproductives.     

Symbiotic "Archaezoa" of the primitive termite Mastotermes darwiniensis still play a role in cellulase production

The relictual Mastotermes darwiniensis is one of the world's most destructive termites. Like all phylogenetically basal termites, it possesses protozoa in its hindgut, which are believed to help it digest wood. L. Li, J. Frohlich, P. Pfeiffer, and H. Konig (Eukaryot. Cell 2:1091-1098, 2003) recently cloned the genes encoding cellulases from the protozoa of M. darwiniensis; however, they claimed that these genes are essentially inactive, not contributing significantly to cellulose digestion. Instead, they suggested that the protozoa sequester enzymes produced by the termite in its salivary glands and use these to degrade cellulose in the hindgut. We tested this idea by performing gel filtration of enzymes in extracts of the hindgut, as well as in a combination of the salivary glands, foregut, and midgut. Three major cellulases were found in the hindgut, each of which had a larger molecular size than termite-derived salivary gland enzymes. N-terminal amino acid sequencing of one of the hindgut-derived enzymes showed that it was identical to the putative amino acid sequence of one mRNA sequence isolated by Li et al. (Eukaryot. Cell 2:1091-1098, 2003). The overall activity of the hindgut cellulases was found to be of approximately equal magnitude to the termite-derived cellulases detected in the mixture of salivary gland, foregut, and midguts. Based on these results, we conclude that, contrary to Li et al. (Eukaryot. Cell 2:1091-1098, 2003), the hindgut protozoan fauna of M. darwiniensis actively produce cellulases, which play an important role in cellulose digestion of the host termite.     

16S rDNA sequence and phylogenetic position of an uncultivated spirochete from the hindgut of the termite Mastotermes darwiniensis Froggatt

Abstract We have analyzed the 16S rDNA sequence and the phylogenetic position of an uncultivated spirochete from the hindgut contents of the Australian termite Mastotermes darwiniensis Froggatt. The 16S rRNA genes of bacteria from the hindgut contents of Mastotermes darwiniensis were amplified by polymerase chain reaction. The amplification products were cloned and sequenced. The sequences were compared to known homologous primary structures. Two of the clones (MDS1 and MDS3) had an insert of 1498 nucleotides showing typical signatures of spirochete 16S rRNA sequences. The sequences of the two clones were most similar to the 16S rRNA sequence of Spirochaeta stenostrepta (89.8%) and Treponema sp. strain H1 (90.7%). Phylogenetical analysis positioned the hindgut spirochete sequence with that of the free-living anaerobic Spirochaeta stenostrepta and Treponema sp. strain H1 as its nearest relatives within the cluster of the spirochetes. We conclude that the analyzed SSU rDNA sequences originate from a spirochete related to the genus Treponema . It is possibly one of the uncultivated unique spirochetes symbiotic in termite hindguts.     

In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata

Sulfate-reducing bacteria (SRB) from termites have been assigned to the genus Desulfovibrio. Desulfovibrio intestinalis lives in the gut of the Australian termite Mastotermes darwiniensis. For the first time we were able to enrich and identify a sulfate-reducing bacterium from the gut of the rose-chafer Pachnoda marginata, which showed the highest 16S rDNA sequence identity (93%) to Desulfovibrio intestinalis and Desulfovibrio strain STL1. Compared to Mastotermes darwiniensis (1x10(7) cells of SRB per ml gut contents), sulfate-reducing bacteria occurred in higher numbers in the gut contents of Pachnoda marginata reaching cell titers of up to 2x10(8) cells per ml gut contents. In vitro sulfate reduction rates were determined with SRB from the gut contents of the termite Mastotermes darwiniensis and the beetle Pachnoda marginata. Due to the higher cell titer, the sulfate reduction rate of Pachnoda marginata was 10(4) nmolxh-1xml-1 and therefore, 21 times higher than that of Mastotermes darwiniensis. In addition, we detected in vivo sulfate reduction in Mastotermes darwiniensis, which indicates that sulfate reducers play an active role in the sulfur metabolism in the termite gut.     

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall. Received: 17 May 1999 / Accepted: 16 September 1999     

Angular sensitivity of the bee ommatidium

Summary Light propagation within the worker bee ommatidium is analyzed. The angular sensitivity of the ommatidium can be separated into a product of the bee's dioptric apparatus and rhabdom angular sensitivities. The discrepancy in angular sensitivity between recent electrophysiological measurements and ray tracing procedures for the worker bee arises because the field of view of the rhabdom is smaller than that of the optical system in front of it.I thank G. A. Horridge for suggesting this research, Simon Laughlin for providing the results of his recordings from the worker bee and J. W. Blamey for helpful discussion on optical systems.     

Neuronal differentiation in Drosophila ommatidium

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.     

Cell fate in the Drosophila ommatidium

Lesions in the Drosophila gene sevenless cause the cell normally destined to differentiate as photoreceptor 7 of the ommatidium to become an accessory lens-secreting cell, the equatorial cone cell. In both P-element- and EMS-induced alleles, the developmental transformation occurs identically. The mutation is cell autonomous, showing that the developmental failure is intrinsic to the transformed cell. Histological and immunological analyses indicate that the cell fails to operate any photoreceptor differentiation machinery prior to adopting the cone cell pathway.     

Neuronal differentiation in the Drosophila ommatidium

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.     

The cellulolytic system of the termite gut

The demand for the usage of natural renewable polymeric material is increasing in order to satisfy the future needs for energy and chemical precursors. Important steps in the hydrolysis of polymeric material and bioconversion can be performed by microorganisms. Over about 150 million years, termites have optimized their intestinal polysaccharide-degrading symbiosis. In the ecosystem of the “termite gut,” polysaccharides are degraded from lignocellulose, such as cellulose and hemicelluloses, in 1 day, while lignin is only weakly attacked. The understanding of the principles of cellulose degradation in this natural polymer-degrading ecosystem could be helpful for the improvement of the biotechnological hydrolysis and conversion of cellulose, e.g., in the case of biogas production from natural renewable plant material in biogas plants. This review focuses on the present knowledge of the cellulose degradation in the termite gut.