Modification of arthropod vector competence via symbiotic bacteria

Some of the world's most devastating diseases are transmitted by arthropod vectors. Attempts to control these arthropods are currently being challenged by the widespread appearance of insecticide resistance. It is therefore desirable to develop alternative strategies to complement existing methods of vector control. In this review, Charles Beard, Scott O'Neill, Robert Tesh, Frank Richards and Serap Aksoy present an approach for introducing foreign genes into insects in order to confer refractoriness to vector populations, ie. the inability to transmit disease-causing agents. This approach aims to express foreign anti-parasitic or anti-viral gene products in symbiotic bacteria harbored by insects. The potential use of naturally occurring symbiont-based mechanisms in the spread of such refractory phenotypes is also discussed.

Amino acid synthesis in the symbiotic sea anemone Aiptasia pulchella

Symbiotic Aiptasia pulchella and freshly isolated zooxanthellae were incubated in (NaHCO3)-C-14 and NH4Cl for 1 to 240 min, and samples were analysed by reverse-phase high-performance liquid chromatography (HPLC) and an online radiochemical detector. NH4+ was first assimilated into C-14-glutamate and C-14-glutamine in the zooxanthellae residing in A. pulchella. The specific activities (dpm nmol(-1)) of C-14-glutamate and C-14-glutamine in vivo, were far greater in the zooxanthellae than in the host tissue, indicating that NH4+ was principally incorporated into the glutamate and glutamine pools of the zooxanthellae. C-14-alpha-ketoglutarate was taken up from the medium by intact A. pulchella and assimilated into a small amount of C-14-glutamate in the host tissue, but no C-14-glutamine was detected in the host fraction. The C-14-glutamate that was synthesized was most likely produced from transamination reactions as opposed to the direct assimilation of NH4+. The free aminoacid composition of the host tissue and zooxanthellae of A. pulchella was also measured. The results presented here demonstrate that NH4+ was initially assimilated by the zooxanthellae of A. pulchella.

Rapid isolation of high-quality RNA from symbiotic dinoflagellates

This report details a reliable and efficient RNA extraction protocol for the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (Gymnodiniales, Dinophyceae). The method typically gives yields of 500 mu g total RNA from 0.4 g wet weight of algae, and, in comparison to current protocols, it is technically simple and less time consuming. This method isolates high-quality, intact RNA from in vine cultured as well as host-isolated cells, as demonstrated by spectrophotometry, gel electrophoresis, and northern analysis. The total RNA obtained was suitable for reverse transcription and PCR amplification of Symbiodinium cDNAs. We have successfully applied our method to isolate total RNA from a different dinoflagellate, Amphidinium carterae Hulburt (Gymnodiniales, Dinophyceae), found in symbiotic association with marine invertebrates.

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

Availability of two forms of dissolved nitrogen to the coral Pocillopora damicornis and its symbiotic zooxanthellae

The relative contribution of dissolved nitrogen (ammonium and dissolved free amino acids DFAAs) to the nitrogen budget of the reef-building coral Pocillopora damicornis was assessed for colonies growing on control and ammonium-enriched reefs at One Tree Island (southern Great Barrier Reef) during the ENCORE (Enrichment of Nutrient on Coral Reef; 1993 to 1996) project. P. damicornis acquired ammonium at rates of between 5.1 and 91.8 nmol N cm(-2) h(-1) which were not affected by nutrient treatment except in the case of one morph. In this case, uptake rates decreased from 80.5 to 42.8 nmol cm(-2) h(-1) (P < 0.05) on exposure to elevated ammonium over 12 mo. The presence or absence of light during measurement did not influence the uptake of ammonium ions. Nitrogen budgets revealed that the uptake of ammonium from concentrations of 0.11 to 0.13 mu M could completely satisfy the demand of growing P. damicornis for new nitrogen. P. damicornis also took up DFAAs at rates ranging from 4.9 to 9.8 nmol N cm(-2) h(-1). These rates were higher in the dark than in the light (9.0 vs 5.1 nmol m(-2) h(-1), P < 0.001). Uptake rates were highest for the amino acids serine, arginine and alanine, and lowest for tyrosine. DFAA concentrations within the ENCORE microatolls that received ammonium were undetectable, whereas they ranged up to 100 nM within the control microatolls. The contribution of DFAAs to the nitrogen budget of P. damicornis constituted only a small fraction of the nitrogen potentially contributed by ammonium under field conditions. Even at the highest field concentrations measured during this study, DFAAs could contribute only similar or equal to 11.3% of the nitrogen demand of P. damicornis. This contribution, however, may be an important source of nitrogen when other sources such as ammonium are scarce or during periods when high concentrations of DFAAs become sporadically available (e.g. cell breakage during fish-grazing).

Photoinhibition and photoprotection in symbiotic dinoflagellates from reef-building corals

Pulse-amplitude-modulation fluorometry and oxygen respirometry were used to investigate diel photosynthetic responses by symbiotic dinoflagellates to light levels in summer and winter on a high latitude coral reef. The symbiotic dinoflagellates from 2 species of reef-building coral (Porites cylindrica and Stylophora pistillata) showed photoinhibitory decreases in the ratio of variable (F-v) to maximal (F-m) fluorescence (F-v/F-m) as early as 09:00 h on both summer and winter days on the reefs associated with One Tree Island (23 degrees 30' S, 152 degrees 06' E; Great Barrier Reef, Australia). This was due to decreases in maximum, F-m, and to a smaller extent minimum, F-0, chlorophyll fluorescence. Complete recovery took 4 to 6 h and began to occur as soon as light levels fell each day. Chlorophyll fluorescence quenching analysis of corals measured during the early afternoon revealed classic regulation of photosystem II (PSII) efficiency through non-photochemical quenching (NPQ). These results appear to be similar to data collected for other algae and higher plants, suggesting involvement of the xanthophyll cycle of symbiotic dinoflagellates in regulating the quantum efficiency of PSII. The ability of symbiotic dinoflagellates to develop significant NPQ, however, depended strongly on when the symbiotic dinoflagellates were studied. Whereas symbiotic dinoflagellates from corals in the early afternoon showed a significant capacity to regulate the efficiency of PSII using NPQ, those sampled before sunrise had a slower and much reduced capacity, suggesting that elements of the xanthophyll cycle are suppressed prior to sunrise. A second major finding of this study is that the quantum efficiency of PSII in symbiotic dinoflagellates is strongly diurnal, and is as much as 50% lower just prior to sunrise than later in the day. When combined with oxygen flux data, these results indicate that a greater portion of the electron transport occurring later in the day is likely to be due to the increases in the rate of carbon fixation by Rubisco or to higher flutes through the Mehler-Ascorbate-Peroxidase (MAP) cycle.

Changes in quantum efficiency of Photosystem II of symbiotic dinoflagellates of corals after heat stress, and of bleached corals sampled after the 1998 Great Barrier Reef mass bleaching event Ross J. Jones , Selina Ward, Affendi Yang Amri and Ove Hoegh-Guldberg

Pulse-amplitude-modulation chlorophyll fluorometry was used to examine changes in dark-adapted F-v/F-m of endosymbiotic dinoflagellate microalgae within the tissues of the temperate coral Plesiastrea versipora exposed to elevated seawater temperature. The F-v/F-m was markedly reduced following exposure of corals to 28 degrees C for 48 h. When corals were returned to ambient (24 degrees C) conditions, F-v/F-m increased in an initial rapid and then secondary slower phase. Tissue discolouration (coral bleaching), caused by a significant decrease in the density of algae, was observed during the first 2-3 days of the recovery period. After 14 days, F-v/F-m was still significantly lower than in control corals. The recovery of F-v/F-m is discussed in terms of repair processes within the symbiotic algae, division of healthy algae and also the selective removal of photo-damaged dinoflagellates. Under field conditions, bleached corals sampled at Heron Island Reef during a bleaching event had significantly lower F-v/F-m than non-bleached colonies; four months after the bleaching event, there were no differences in F-v/F-m or algal density in corals marked as having bleached or having shown no signs of colour loss. The results of this laboratory and field study are consistent with the hypothesis that an impairment of photosynthesis occurs during heat-stress, and is the underlying cause of coral bleaching.

Aspergillicins A-E: five novel depsipeptides from the marine-derived fungus Aspergillus carneus

A search for new antiparasitic agents from a strain of the fungus Aspergillus carneus isolated from an estuarine sediment collected in Tasmania, Australia, yielded the known terrestrial fungal metabolite marcfortine A ( 1) as an exceptionally potent antiparasitic agent. This study also yielded a series of new depsipeptides, aspergillicins A - E ( 2 - 6) and the known terrestrial fungal metabolite acyl aszonalenin ( 7). Marcfortine A ( 1) and acyl aszonalenin ( 7) were identified by spectroscopic analysis, with comparison to literature data. Complete stereostructures were assigned to aspergillicins A - E ( 2 - 6) on the basis of detailed spectroscopic analysis, together with ESIMS analysis of the free amino acids generated by acid hydrolysis, and HPLC analysis of Marfey derivatives prepared from the acid hydrolysate. The peptide amino acid sequence for all aspergillicins was unambiguously assigned by MSn ion-trap ESI mass spectrometry.

Nutrient-induced perturbations to delta C-13 and delta N-15 in symbiotic dinoilagellates and their coral hosts

Inorganic nutrients play a critical role in determining benthic community structure in tropical seas. This study examined the impact of adding inorganic nutrients (ammonium and phosphate) on the isotopic composition of 2 reef-building corals, Pocillopora damicornis and Heliofungia actiniformis, on the southern Great Barrier Reef. The addition of elevated nutrients to patch reefs that pond at low tide did not perturb the C:N ratio of either species or their symbiotic dinoflagellates. The C:N ratios were significantly higher in material extracted from the skeleton (14.8 +/- 1.50 and 10.8 +/- 1.42) than either host (7.6 +/- 0.87 and 6.0 +/- 0.71) or symbiotic dinoflagellates (5.7 +/- 0.48 and 6.9 +/- 0.66) (P. damicornis and H. actiniformis respectively; 95 confidence intervals). The ratio of acquired N to background N suggests that the added dissolved inorganic nitrogen (DIN) accounted for 50 to 100% of total nitrogen within the tissues of P. damicornis and H. actiniformis at the end of the experiment. The addition of the isotopically depleted nutrients (delta(15) N = 0parts per thousand) to patch reefs significantly decreased delta(15)N from control values of between 3 and 4 to values to below 1 in the case of all compartments, while delta(13)C values were relatively unresponsive to nutrient treatments. These findings suggest that coral delta(15)N has the potential to provide a historical record of the delta(15)N of dissolved nitrogen surrounding reef-building corals and their symbiotic dinoflagellates.

Genetic variability of the symbiotic dinoflagellates from the wide ranging coral species Seriatopora hystrix and Acropora longicyathus in the Indo-West Pacific

The scleractinian coral species, Seriatopora hystrix and Acropora longicyathus, are widely distributed throughout the latitudinal range of the tropical west Pacific. These 2 coral species live in a mutually beneficial relation with symbiotic dinoflagellates (zooxanthellae), which are passed to their progeny by vertical transmission (zooxanthellate eggs or larvae) and horizontal transmission (eggs or larvae that acquire symbionts from the environment), respectively. For S. hystrix, vertical transmission might create biogeographically isolated and genetically differentiated symbiont populations because the extent of its larval migration is known to be limited. On the other hand, horizontal transmission in corals such as A. longicyathus may result in genetically connected symbiont populations, especially if its zooxanthellae taxa are widely distributed. To examine these hypotheses, symbionts were collected from colonies of S. hystrix and A. longicyathus living in the Great Barrier Reef (Australia), South China Sea (Malaysia) and East China Sea (Ryukyus Archipelago, Japan), and were examined using restriction fragment length polymorphism and sequence analysis of large and small subunit rRNA genes. Phylogenetic analysis assigned the symbionts to 1 of 3 taxonomically distinct groups, known as clades. Symbionts from Australian and Japanese S. hystrix were placed in Clade C, and Malaysian S. hystrix symbionts in the newly described Clade D. Seven of 11 Australian and all Japanese and Malaysian colonies of A. longicyathus had symbiotic dinoflagellates that also grouped with Clade C, but symbionts from the remaining Australian colonies of A. longicyathus grouped with Clade A. Analysis of molecular variance of Clade C symbionts found significant genetic variation in 1 or more geographic groups (69.8%) and to a lesser extent among populations within geographic regions (13.6%). All populations of Clade C symbionts from S. hystrix were genetically differentiated according to geographic region. Although Clade C symbionts of A. longicyathus from Japan resolved into a distinct geographic group, those from Australia and Malaysia did not and were genetically connected. We propose that these patterns of genetic connectivity correlate with differences in the dispersal range of the coral or symbiont propagules and are associated with their respective modes of symbiont transmission.

Diurnal changes in the photochemical efficiency of the symbiotic dinoflagellates (Dinophyceae) of corals: photoprotection, photoinactivation and the relationship to coral bleaching

The photochemical efficiency of symbiotic dinoflagellates within the tissues of two reef-building corals in response to normal and excess irradiance at wafer temperatures < 30 C were investigated using pulse amplitude modulated (PAM) chlorophyll fluorescence techniques, Dark-adapted F-v/F-m showed clear diurnal changes, decreasing to a low at solar noon and increasing in the afternoon. However, F-v/F-m also drifted downwards at night or in prolonged darkness, and increased rapidly during the early morning twilight. This parameter also increased when the oxygen concentration of the wafer holding the corals was increased. Such changes have not been described previously, and most probably reflect state transition's associated with PQ pool reduction via chlororespiration. These unusual characteristics may be a feature of an endosymbiotic environment, reflective of the well-documented night-time tissue hypoxia that occurs in corals. F-v/F-m decreased to 0.25 in response to full sunlight in shade-acclimated (shade) colonies of Stylophora pistillata, which is considerably lower than in light-acclimated (sun) colonies. In sun colonies, the reversible decrease in F-v/F-m was caused by a lowering of F-m and F-o suggesting photoprotection and no lasting damage. The decrease in F-v/F-m, however, was caused by a decrease in F-m and an increase in F-o in shade colonies suggesting photoinactivation and long-term cumulative photoinhibition. Shade colonies rapidly lost their symbiotic algae (bleached) during exposure to full sunlight. This study is consistent with the hypothesis that excess light leads to chronic damage of symbiotic dinoflagellates and their eventual removal from reef-building corals. It is significant that this can occur with high light conditions alone.

Pseudotrypanosoma elphinstonae sp n., a trichomonad symbiotic in Schedorhinotermes (Isoptera : Rhinotermitidae)

A new species of Pseudotrypanosoma, P. elphinstonae sp. n., is described which is symbiotic within the hindguts of the rhinotermitid termites Schedorhinotermes secundus and S. intermedius. P. elphinstonae possesses most of the features of the genus: 4 anterior flagella, prominent costa and recurrent flagellum forming an undulating membrane and simple bean-shaped parabasal body. The mastigont complex is of similar composition and arrangement to other trichomonads but the pelto-axostylar complex is greatly simplified being composed of a single layer of microtubules which do not over lap and cannot be distinguished into separate structures. The undulating membrane is much smaller than in related species; the costa is smaller and simpler and there are no microtubular bundles connecting it to the recurrent flagellum. Comparison of the ultrastructure of P. elphinstonae sp. n. with that of P. giganteum demonstrated that P. elphinstonae sp. n. in addition to being much smaller in size had a correspondingly simper ultrastructural organisation lacking several organelles which characterise the latter species.