Development of a tissue cell culture bioassay for identifying mechanisms of inhibition of settlement of barnacle and tunicate larvae by surface‐colonizing marine bacteria

A marine bacterium D2 (CCUG 26757) isolated from a tunicate Ciona intestinalis specimen produced a low molecular weight component which inhibits barnacle and tunicate larvae and prevents their settlement on solid surfaces (Holmström et al., 1992). In order to perform chemical and structural analyses of the component independent of season, a bioassay, complementary to tests with invertebrate larvae was developed. This bioassay is based on tissue cell culture techniques and growth of the AGS cell line. It was previously shown that the toxic component is a stationary phase released product, which is heat stable and less than 500 Dalton in size (Holmström et al., 1992). Furthermore, it is not a peptide or a protein and metaperiodate treatment increases its toxicity to larvae indicating that it binds to or contains carbohydrate moieties. In this study, these results were confirmed by using the anchorage dependent human gastric adenocarcinoma (AGS) cell line as a bioassay. Fractionation of the D2 supernatant on a Sephadex G‐200 column and addition of different size fractions to the AGS tissue culture cells, showed that both a low and a high molecular weight fraction inhibited cell growth. Exposure of Balanus amphitrite and C. intestinalis larvae to the same fractions, showed that the low molecular weight fraction that inhibited growth of the cells corresponds to the component that inhibited larvae of both organisms. The high molecular weight fraction was found to inhibit larvae of B. amphitrite.     

Inhibition of common fouling organisms by marine bacterial isolates ith special reference to the role of pigmented bacteria

Two questions of relevance to the establishment of marine biofouling communities were addressed, viz (1) what is the frequency with which bacterial strains isolated from living and inanimate surfaces in the marine environment show inhibitory activity against the settlement of common fouling organisms, and (2) is the antifouling bacterium, D2, an inhabitant of different marine waters, and how unique is this bacterium, in its mode of action against different target organisms? With respect to the first question, ninety three marine bacteria isolated from various rock surfaces from the marine environment were tested against larvae of Balanus amphitrite and spores of Ulva lactuca. Settlement assays against the diatom Amphora sp. were also performed on 10 of these strains. Nine bacterial isolates were shown to be inhibitory against larval settlement and eight of these strains were also inhibitory against algal spores. Altogether 16 strains were inhibitory against the settlement of algal spores while none of the bacterial strains inhibited diatom settlement. With respect to the second question, D2, a dark green pigmented bacterium, isolated from an adult tunicate off the Swedish west coast, has been found to be a very effective inhibitor against common fouling organisms. In order to see if this bacterium can be found in other marine waters, bacteria from living surfaces of marine plants and animals from waters around Sydney, Australia, were isolated and screened for inhibitory activity against barnacle larvae. Seventy four percent of the 23 plant isolates were shown to be inhibitory against larval settlement while only 30% of the 23 isolates from marine animals reduced settlement. Twenty two of the isolates from different seaweeds were dark pigmented and 20 of these strains inhibited settlement of barnacle larvae and algal spores. Three of the strains showed the same phenotypic expression as D2, and the results indicate that these strains may be D2 or closely related strains, suggesting that D2 may be a common inhabitant in the marine environment.     

The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae

Comparative bacteriological and serological studies of three isolates and the reference strain of Bacillus thuringiensis subsp. darmstadiensis (serotype 10) were conducted. No difference was shown in the flagellar antigenic structure between the three isolates and the reference strain. Differences were observed in the O antigenic structures and in the following biochemical properties: lecithinase production, DNase production, arginine decarboxylase production, acid production from inulin, and malonate utilization. β-Exotoxin production was not detected in these three isolates. The reference strain produced parasporal inclusions toxic to the lepidopterous larvae but nontoxic to mosquito larvae. On the contrary, two among the three isolates, which produced spherical parasporal inclusions, were not toxic to the lepidopterous larvae but highly toxic to larvae of the mosquitoes, Culex tritaenlorhynchus, Culex molestus, and Aedes aegypti. Another isolate produced large irregular-shaped inclusions nontoxic to the insects of both orders. Accordingly, B. thuringiensis serotype 10 was divided into three groups from the viewpoint of toxicity against lepidopterous and mosquito larvae.     

Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.     

Identity of a microsporidium from three New Zealand pasture insects: Costelytra zealandica (Coleoptera,scarabeidae), Wiseana spp. (Lepidoptera,Hepialidae), and Listronotus bonariensis (Coleoptera,Curculionidae)

The life cycles of multisporous microsporidia isolated from grass grubs (Costelytra zealandica), porina (Wiseana spp.), and Argentine stem weevils (Listronotus bonariensis) are investigated by light and electron microscopic examination of laboratory-reared and -infected specimens. Each microsporidian isolate is cross-infectious to each host insect, as well as to laboratory-reared larvae of Planotortrix excessana (Lepidoptera, Tortricidae). Comparison with live material of Pleistophora oncoperae (Microsporida, Pleistophoridae), collected from Oncopera alboguttata (Lepidoptera, Hepialidae) in Australia, indicates that these isolates are synonymous with P. oncoperae. Ultrastructural features suggest that this species should be transferred to the genus Vavraia.     

Fermentation media and production of exotoxin by three varieties of Bacillus thuringiensis

The insecticidal activities of the exotoxin produced by three varieties of Bacillus thuringiensis grown in six fermentation media were determined by testing the supernatants against larvae of the house fly, Musca domestica, and the black cutworm, Agrotis ipsilon. The activities of the exotoxins from the isolates varied when they were grown in the same medium and also when they were grown in different media. When an isolate of B. thuringiensis var. thuringiensis, and of var. tolworthi were grown in proflo broth, the supernatants produced were more toxic to house fly than to black cutworm larvae, indicating the presence of more than one exotoxin. Autoclaving the supernatants for 15 and 30 min further demonstrated the presence of several exotoxins.     

Screening for mycotoxins with larvae of Tenebrio molitor.

Larvae of the yellow mealworm, Tenebrio molitor, were used to screen isolates of field and storage fungi, included in their dietary substrate, for mycotoxins. Feeding of three isolates of Fusarium roseum, two of Fusarium equiseti and of Fusarium nivale, and one of an unidentified species of Fusarium resulted in growth depression of the larvae. One isolate of an unidentified species of Myrothecium was also toxic to larvae of Tenebrio molitor. Mycotoxin production was apparently dependent, not only on the fungal isolate, but also on the culture conditions under which the fungus was grown. Some fungal isolates had growth-promoting qualities for larvae of Tenebrio molitor.     

Comparative studies of eleven isolates of the fungal entomopathogen Tolypocladium cylindrosporum and two isolates of Tolypocladium extinguens

The virulence of 11 isolates of Tolypocladium cylindrosporum and 2 isolates of Tolypocladium extinguens was evaluated against mosquito larvae. Second-instar Aedes aegypti larvae were more susceptible to infection by blastospores than were second-instar Anopheles stephensi larvae. Only the T. extinguens isolates were not infectious for A. aegypti or A. stephensi. Nine of the remaining strains killed 100% of larvae at the highest dose tested (10⁶ blastospores/ml). In vitro sporulation and conidiospore size were also studied. Significant differences were observed for both characteristics. Characterization of strains according to in vitro sporulation and virulence made it possible to select the most promising strains for future laboratory and field tests against mosquitoes.     

Morphological Differences between Larvae of the Ciona intestinalis Species Complex: Hints for a Valid Taxonomic Definition of Distinct Species

The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.     

Effects of infection of EGFP-expressing Escherichia coli on haemocytes in Ciona intestinalis

The effects of infection of EGFP-expressing Escherichia coli on the haemocytes of the ascidian Ciona intestinalis were investigated. The results showed that THC of the infected individuals changed significantly. Hyaline amoebocytes phagocytosed E. coli in 5 min and excreted lysosome particles that attached to the surface of the bacteria. Granular amoebocytes released lots of particles for humoral immunity while stem-cell-like haemocytes remained intact. With the increase in THC, the stem-cell-like haemocytes showed division and proliferation. A small portion of hyaline amoebocytes was at early apoptosis stage 1 h after infection and typical apoptosis bodies emerged in granular amoebocytes. A few of the infected haemocytes showed DNA damage using SCGE assay. Flow cytometry analysis revealed an obvious apoptosis peak in infected haemocytes. In conclusion, apoptosis was found to be an important immune response of ascidian haemocytes response to bacterial infection. To our best knowledge, this is the first report of the occurrence of apoptosis of haemocytes in ascidians.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Proteomic profiles of embryonic development in the ascidian Ciona intestinalis

We report here proteomics-based protein profiles of three embryonic stages of the ascidian Ciona intestinalis. Two-dimensional gel electrophoresis revealed 416, 539, and 695 protein spots in the unfertilized eggs, 16 cell-stage embryos, and tadpole larvae, respectively. Comparative and quantitative analyses of the spot patterns identified proteins showing an increase or decrease in amount during embryonic development. Protein identification by MALDI-TOF/MS indicated not only the abundance and importance of metabolic enzymes and translation elongation factors but also the functional importance of actin-binding proteins and molecular chaperones during ascidian development. Global changes in spots for vitellogenin-like protein suggested post-translational modification or proteolytic digestion of this protein during embryogenesis. Comparison between mRNA and protein levels among unfertilized eggs, 16 cell-stage embryos and tadpole larvae indicated nonparallel expression patterns of genes and proteins. Ascidians provide an excellent system for studying gene expression and cell differentiation during development, and the present study should shed light on the associated molecular mechanism at the protein level.     

Mortality of Delia floralis, Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi

Abstract:  The susceptibility of Delia floralis eggs, neonates and larvae and the susceptibility of Galleria mellonella and Mamestra brassicae larvae to seven different Norwegian isolates of the insect pathogenic, hyphomycetous fungi Tolypocladium cylindrosporum , Metarhizium anisopliae and Beauveria bassiana , were investigated. Metarhizium anisopliae isolate ARSEF 5520 was highly virulent to G. mellonella larvae and caused 100% mortality when tested at a concentration of 3.6 × 10⁶ conidia/ml. The same M. anisopliae isolate was not virulent to D. floralis larvae. Isolates of T.cylindrosporum , were equally virulent to G. mellonella and D. floralis causing up to 36.0% mortality of larvae. It is suspected, however, that the use of grated rutabaga as a food source in the D. floralis bioassay reduced the fungal virulence of both M. anisopliae and T. cylindrosporum to D. floralis . Among three T. cylindrosporum isolates tested at a concentration of 1.0 × 10⁷ conidia/ml against eggs of D. floralis none of them reduced the hatching percentage. One isolate, ARSEF 5525 did, however, significantly reduce the longevity of neonates. Beauveria bassiana isolates ARSEF 5510 and ARSEF 5370 tested at a concentration of 1.0 × 10⁷ conidia/ml resulted in M. brassicae mortality levels of 70.0 and 55.0%, respectively. The B. bassiana isolate ARSEF 5557, however, was not virulent to M. brassicae . Among the three isolates tested against M. brassicae the two virulent isolates produced a red pigment, probably oosporein, when cultured in Sabouraud dextrose agar.     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.     

Assessment of toxic potential of local Jordanian Bacillus thuringiensis strains on Drosophila melanogaster and Culex sp. (Diptera)

AIMS: To assess the toxic potential of different local Jordanian Bacillus thuringiensis isolates on larvae of Drosophila melanogaster and Culex sp. METHODS AND RESULTS: Scanning electron microscopy revealed the presence of spherical, bi-pyramidal, and bi-pyramidal and cuboidal parasporal bodies produced by the toxic isolates. Spherical inclusions dominated. The toxicity of the isolates to the two insects, determined using 24-well plates or vials, indicated that the 50% lethal concentration (LC50) of the bacterial suspension for D. melanogaster and Culex sp. larvae varied from 4.60 to 8.65, and from 5.30 to 6.74, respectively. CONCLUSION: Comparison of the LC50 values of isolate 82 with those of the reference strain B. t. israelensis showed that this isolate has a higher toxicity potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Some local Jordanian B. thuringiensis isolates exhibit toxic potential that could be used to control some important pests, and could replace chemical pesticides.     

The impact of waterborne cues from conspecifics and other species on the larvae of Halichondria panacea Pallas, 1766 (Porifera: Demospongiae)

The impacts of different concentrations of the excretory-secretory products (ESPs) of the solitary ascidian Styela rustica (Linnaeus, 1767) and the sponge Halichondria panacea (Pallas, 1766) on the settlement, metamorphosis, and mortality rates of H. panacea larvae were studied in a laboratory experiment. At high concentrations, substances released into the environment by the ascidian S. rustica exerted a negative impact on the metamorphosis rate of sponge larvae. The exposure to moderate or high concentrations of ESPs from conspecific adults led to high mortality of larval sponges; however, low conspecific ESP concentrations markedly stimulated metamorphosis; larval mortality was low. Apparently, different concentrations of the same ESPs can have effects with a different strength and focus. This should be taken into account in the study of chemically mediated interactions between aquatic organisms.     

Biocontrol evaluation of extracts and a major component,clusianone, from Clusia fluminensis Planch. & Triana against Aedes aegypti

Studies evaluated the effects of hexanic extracts from the fruits and flowers ofClusia fluminensis and the main component of the flower extract, a purified benzophenone (clusianone), against Aedes aegypti. The treatment of larvae with the crude fruit or flower extracts from C. fluminensis did not affect the survival ofAe. aegypti (50 mg/L), however, the flower extracts significantly delayed development of Ae. aegypti. In contrast, the clusianone (50 mg/L) isolate from the flower extract, representing 54.85% of this sample composition, showed a highly significant inhibition of survival, killing 93.3% of the larvae and completely blocking development of Ae. aegypti. The results showed, for the first time, high activity of clusianone against Ae. aegypti that both killed and inhibited mosquito development. Therefore, clusianone has potential for development as a biopesticide for controlling insect vectors of tropical diseases. Future work will elucidate the mode of action of clusianone isolated from C. fluminensis.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus

Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of X. nematophilus subsp. dutki (isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of G. mellonella larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with X. nematophilus was stable at 60°C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27°C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of X. nematophilus. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of X. nematophilus and the time of sampling.