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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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     

The ommatidium of the termite mastotermes darwiniensis

An electron microscope study of the compound eye of the termite Mastotermes darwiniensis shows that the ommatidia have both primitive and specialized features. The ommatidia are of the apposition type with eight similar retinular cells, a fused rhabdom, irregularly orientated rhabdomere tubules and no tracheae. The retinula cells contain lipid. The cone cells have unusual processes, which thicken towards the basement membrane and contain rough endoplasmic reticulum, granules and dense bodies. These processes may act as a primitive transport system for nutrients.     

Morphology of the salivary glands of Mastotermes darwiniensis Froggatt (Isoptera : Mastotermitidae)

The salivary glands of Mastotermes darwiniensis (Isoptera : Mastotermitidae) workers consist of acini made up of peripherally and centrally placed cells with storage cells. A network of small efferent ductules is associated with each acinus. Intracellular ductules of the central and peripheral cells are filled with protruding microvilli. Central cells contain an abundance of rough endoplasmic reticulum and vacuoles. Continua of rough endoplasmic reticulum and vacuoles are present. Pinocytosis does not appear to be involved in secretion by the central cells. Peripheral cells are compartmental, having electron-lucent vesicle accumulations away from intracellular ductules and the basal plasmalemma invaginations. Peripheral cell morphology is similar to cells which are associated with fluid movement. Storage cells contain a large vacuole and exhibit micropinocytosis. Two axon profile types are present near and on the acini and salivary ducts, one of which appears associated with neuronal end plates.     

Symbiotic nitrogen fixation in the New Zealand dampwood termite (

This study investigates symbiotic microorganisms in the New Zealand dampwood termite Stolotermes ruficeps using culture-independent techniques to describe the diversity of nitrogen-fixing organisms within this termite. Phylogenetic analysis of a portion of the nifH gene (encoding dinitrogenase reductase) revealed 19 phylotypes (>98% sequence identity) with 77?86% similarity to published nucleotide sequences from uncultured microorganisms described from termite guts. The majority of sequences obtained in this study were most closely related to sequences obtained from basal families Kalotermitidae, Termopsidae and the closely related wood-feeding cockroach species Cryptocercus. This adds to the growing amount of evidence suggesting that the composition of nifH sequences is characteristic of a termite family. This study also identifies wood-dwelling termites as a potentially important source of nitrogen input into temperate forests, something previously neglected and warranting further investigation.     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termi...     

Components of cellulase from the higher termite,Nasutitermes walkeri

The cellulase from the termite Nasutitermes walkeri consists of two enzymes. Each has broad specificity with predominantly one activity. One enzyme is an endo-gb-1,4-glucanase (EC 3.2.1.4) which predominantly cleaves cellulose randomly to glucose, cellobiose and cellotriose. It hydrolyses cellotetraose to cellobiose but will not hydrolyse cellobiose or cellotriose. The second enzyme component is a β-1,4-glucosidase (EC 3.2.1.21) as its major activity is to hydrolyse cellobiose, cellotriose and cellotetraose to glucose; it has some exoglucosidase activity as glucose is the only product produced from cellulose. Its cellobiase activity is inhibited by glucono-δ-lactone.     

Purification and characterization of cellulase fromBacillus thermoalcaliphilus isolated from a termite mound

An extracellular carboxymethyl cellulase (CMCase) was purified to homogeneity fromBacillus thermoalcaliphilus sp. nov. The cellulase was composed of a single subunit with molar mass of 46 kDa. The apparentK _m was at 3.5 mg cellulose per mL. Its optimum pH was 8.5, it was most stable at pH 8.5–9.5 but 50% of enzyme activity was present after 30 min at pH 11.0. The activity was highest at 70°C.     

Thermoregulation of termite mounds: what role does ambient temperature and metabolism of the colony play?

Summary: Termites are well known for their ability to regulate the environment of their nest such as temperature and humidity. The influence of fluctuating ambient temperature and mound characteristics on mean nest temperature and daily fluctuation of nest temperature was analysed quantitatively in the fungus-cultivating, mound-building termite Macrotermes bellicosus (Macrotermitinae) in the savanna of the Comoé National Park (Côte d'Ivoire). Additionally, the nest temperatures of inhabited and uninhabited mounds were compared to analyse the contribution of ambient temperature to nest temperature in relation to metabolic heat production of the termites and their fungi. Mound structure alone resulted in a relatively constant nest temperature. Abiotic heat production via solar radiation alone yielded nest temperatures that corresponded to mean ambient temperatures. However, only the production of metabolic heat by the termites and the fungi increased these temperatures to the actual nest temperature. Therefore, and due to the high heat capacities of the mounds, large colonies (mound height above 2.0 m) had higher nest temperatures than smaller ones. Only large colonies attain constant nest temperatures of 30 °C that are largely independent from ambient temperatures and optimal for the growth and development of the termites and their fungi.