Decreased toxicity of Bacillus thuringiensis subsp. israelensis to mosquito larvae after contact with leaf litter

Bacillus thuringiensis subsp. israelensis is a bacterium producing crystals containing Cry and Cyt proteins, which are toxic for mosquito larvae. Nothing is known about the interaction between crystal toxins and decaying leaf litter, which is a major component of several mosquito breeding sites and represents an important food source. In the present work, we investigated the behavior of B. thuringiensis subsp. israelensis toxic crystals sprayed on leaf litter. In the presence of leaf litter, a 60% decrease in the amount of Cyt toxin detectable by immunology (enzyme-linked immunosorbent assays [ELISAs]) was observed, while the respective proportions of Cry toxins were not affected. The toxicity of Cry toxins toward Aedes aegypti larvae was not affected by leaf litter, while the synergistic effect of Cyt toxins on all B. thuringiensis subsp. israelensis Cry toxins was decreased by about 20% when mixed with leaf litter. The toxicity of two commercial B. thuringiensis subsp. israelensis strains (VectoBac WG and VectoBac 12AS) and a laboratory-produced B. thuringiensis subsp. israelensis strain decreased by about 70% when mixed with leaf litter. Taken together, these results suggest that Cyt toxins interact with leaf litter, resulting in a decreased toxicity of B. thuringiensis subsp. israelensis in litter-rich environments and thereby dramatically reducing the efficiency of mosquitocidal treatments.     

Comparative ability to detoxify alder leaf litter in field larval mosquito collections

The larvicidal effects of polyphenols from dietary alder leaf litter were investigated in different field collections of three detritivorous Aedes taxa (Ae. detritus, Ae. cataphylla, Ae. rusticus) and compared to the cytochrome P450 monooxygenase, glutathione S-transferase, and esterase activities. Larvae from polyphenol-rich habitats had a higher tolerance for polyphenols and higher midgut cytochrome P450 and esterase activities than larvae from polyphenol-poor habitats. Furthermore, the role of P450 enzymes in the mechanism of resistance to alder polyphenols was suggested by the synergistic effect in vivo of piperonyl butoxide in the resistant Ae. rusticus. This confirms the importance of polyphenols to larval mosquito performance, and provides evidence for the importance of specific detoxification mechanisms for tolerance to dietary polyphenols. Arch.     

Serological classification of Bacillus sphaericus strains on the basis of toxicity to mosquito larvae

Summary A toxicity study of 54 Bacillus sphaericus strains isolated from vectors or breeding sites has led to a relatively homogeneous grouping of mosquito pathogenic strains into five H-serotypes among the nine serotypes determined. Each serotype seems to be characterized by a different level of toxicity and a classification of these five serotypes can be made on the basis of this toxicity. Within these serotypes, a scale of toxicity has been tentatively fixed and an arbitrary limit of toxicity suggested.     

Electrical hypothesis of toxicity of the Cry toxins for mosquito larvae

Many electrical properties of insect larval guts have been studied, but their importance for toxicity of the Cry-type toxins has never been reported in the literature. In the present work, we observed potential-dependent permeabilization of plasma membrane by several polycationic peptides derived from the Cry11Bb protoxin. The peptide BTM-P1d, all D-type amino acid analogue of the earlier reported peptide BTM-P1, demonstrated high membrane-permeabilizing activity in experiments with isolated rat liver mitochondria, RBC (red blood cells) and mitochondria in homogenates of Aedes aegypti larval guts. Two larger peptides, BTM-P2 and BTM-P3, as well as the Cry11Bb protoxin treated with the protease extract of mosquito larval guts showed similar effects. Only protease-resistant BTM-P1d, in comparison with other peptides, displayed A. aegypti larval toxicity. Taking into account the potential-dependent mechanism of membrane permeabilization by studied fragments of the Cry11Bb protoxin and the literature data related to the distribution of membrane and transepithelial potentials in the A. aegypti larval midgut, we suggest an electrical hypothesis of toxicity of the Cry toxins for mosquito larvae. According to this hypothesis, the electrical field distribution is one of the factors determining the midgut region most susceptible for insertion of activated toxins into the plasma membrane to form pores. In addition, potential-dependent penetration of short active toxin fragments into the epithelial cells could induce permeabilization of mitochondria and subsequent apoptosis or necrosis.     

Differential palatability of leaf litter to four sympatric isopods in a Hong Kong forest

Summary The feeding behaviour of four sympatric isopods from a Hong Kong forest has been investigated. The study included two armadillids (Formosillo raffaelei and Orodillo maculatus) and two philosciids (Burmoniscus ocellatus and Burmoniscus sp.). When given a choice of eight types of litter, all isopod species showed significant dietary selection, and food preferences were similar. Berchemia racemosa (Rhamnaceae) was most readily consumed, followed by Celtis sinensis (Ulmaceac), while feeding rates on Cinnamomum camphora (Lauraceae) were low. The armadillids exhibited a narrower dietary spectrum than the philosciids. All isopods showed significant differences in consumption rates when each litter type was presented separately to them. Cinnamomum camphora was eaten the slowest, while Celtis sinensis and Berchemia racemosa were the top-ranked species. Although patterns of litter ranking based on percentage eaten or feeding rates were similar, all isopods ate more food when given a mixture of leaves than when presented with a single litter type. Assimilation rates were, in general, positively related to feeding rates. On this basis, it appears that assimilation may influence food preference. There were no clear relationships between food preference or feeding rates and ash, calcium, copper, soluble tannin or energy content of the litter. This finding may indicate the benefits of isopods maintaining a mixed diet, consuming certain litter species to meet their calcium or copper requirements and then switching to others so as to meet daily energy needs and to avoid excessive injection of tannins or plant allelochemicals.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

水生双翅目昆虫监测水体重金属污染的研究

水生双翅目昆虫是监测水体重金属污染的理想对象。文章归纳用于监测重金属污染的水生双翅目昆虫的种类,重点介绍水生双翅目昆虫在重金属污染下外部形态、内部结构、生化及分子水平的变化,以及相关生物标志物的研究,为水生双翅目昆虫用于水体重金属污染的生物监测提供科学依据。     

Differential responses of ammonia/ammonium-oxidizing microorganisms in mangrove sediment to amendment of acetate and leaf litter

The effects of acetate and leaf litter powder on ammonia/ammonium-oxidizing microorganisms (AOMs) in mangrove sediment were investigated in a laboratory incubation study for a period of 60 days. The results showed that different AOMs responded differently to the addition of acetate and leaf litter. A higher diversity of anaerobic ammonium-oxidizing (anammox) bacteria was observed when acetate or leaf litter was added than the control. However, acetate and leaf litter generally inhibited the growth of anammox bacteria despite that leaf litter promoted their growth in the first 5 days. The inhibitory effects on anammox bacteria were more pronounced by acetate than by leaf litter. Neither acetate nor leaf litter affected ammonia-oxidizing archaea (AOA) community structures, but promoted their growth. For ammonia-oxidizing bacteria (AOB), the addition of acetate or leaf litter resulted in changes of community structures and promoted their growth in the early phase of the incubation. In addition, the promoting effects by leaf litter on AOB growth were more obvious than acetate. These results indicated that organic substances affect AOM community structures and abundances. The study suggests that leaf litter has an important influence on the community structures and abundances of AOMs in mangrove sediment and affects the nitrogen cycle in such ecosystem.     

Effect of leaf litter and density on fitness and population performance of the hole mosquito Aedes triseriatus

Abstract.

• 1 We tested the effects of food, in the form of leaf litter, and density on population growth and fitness correlates of the tree hole mosquito Aedes triseriatus. Our field experiment used a 2 x 2 randomized block design, with three holes as blocks. In cages within three holes, we manipulated densities of Aedes triseriatus larvae, and presence of leaf litter. Our laboratory experiment used a 2 x 3 factorial design in which we also manipulated densities of larvae and availability of leaf litter within similar cages (inside, outside, or absent). For both experiments we determined effects on survival, days to and mass at eclosion of both sexes, and a composite index of population performance λ′, that estimates finite rate of increase.
• 2 In the field experiment, we found significant and large effects of leaf litter and of density on the performance of A. triseriatus. The effect of density did not depend on leaf litter availability. We documented significant variation among tree holes for most correlates of fitness. Often, the effects of treatments varied significantly depending on the tree hole tested.
• 3 In the laboratory experiment we found significant effects of density and litter, and that direct browsing on leaf litter is necessary for the effect of litter on performance of A. triseriatus. In the laboratory, the negative effect of increased density was dependent on leaf litter availability.
• 4 Our results demonstrate that leaf litter has important effects on population performance of tree hole mosquitoes in natural tree holes. Direct browsing on the surface of leaf litter is the most likely mechanism by which litter enhances population growth. The impact of litter on populations varies among tree holes.

     

Effects of leaf litter species on macroinvertebrate community properties and mosquito yield in Neotropical tree hole microcosms

Detritus quality and quantity affect macroinvertebrate productivity and distribution in many freshwater ecosystems. This study experimentally investigated the effects of leaf litter from Ceiba pentandra, Dipteryx panamensis, Ficus yoponensis, and Platypodium elegans on macroinvertebrate species composition, richness, and abundance in artificial water-filled tree holes in a lowland moist forest of Panama. Species composition was similar among treatments, but species richness and longevity differed among litter types and were consistently highest with Platypodium litter. Similar patterns were observed in natural tree holes of the focal tree species. The mosquito Culex mollis was the most abundant species in the field experiment. Average conductivity and dissolved oxygen concentration differed among leaf species, but pH did not. Leaf toughness was positively correlated with mean macroinvertebrate abundance and cumulative species richness. A laboratory experiment measured C. mollis yield and pupation time in tree hole microcosms containing the four litter species. Cumulative mosquito mass and time to pupation differed among leaf litter species, with Platypodium litter supporting the greatest yield. Pupation was slowest on Ceiba litter. Grazing by mosquito larvae facilitated leaf decomposition in all treatments. Results suggest that differences in macroinvertebrate species richness and mosquito yield can be attributed to differences in nutritional quality among litter species. Received: 14 October 1998 / Accepted: 21 February 1999     

Sporulation of Bacillus sphaericus 2297: an electron microscope study of crystal-like inclusion biogenesis and toxicity to mosquito larvae

Sporulation of Bacillus sphaericus strain 2297 in a synchronous liquid culture was studied by electron microscopy. The t0 of sporulation occurred 7 h after the beginning of the lag phase. Crystal-like inclusions first appeared at t2 and reached their final size between t5 and t6. The release of the spore/inclusion complex occurred at about t15 (22 h after inoculation). Toxicity against Culex pipiens larvae was related to sporulation and appeared during the early stages of sporulation. The LC50 (24 h) decreased about 10(5)-fold between t0-2 and t7, in correlation with the formation of crystalline inclusions. Heat resistance of spores appeared later than toxicity.     

The alarm reaction of mosquito larvae

When Aedes larvae are alarmed they swim actively to the bottom; they recover by floating passively upwards. Culex and Anopheles larvae sink passively when alarmed; they swim actively to the surface on recovery. Repeated stimulation produces stimulus satiation, but a different type of stimulus can then give a complete response. Geotaxis and phototaxis are concerned in the alarm reaction in Aedes; phototaxis can overrule geotaxis. The chill coma temperature of these larvae is several degrees cooler than that at which the alarm reaction takes place. Acclimatization can alter the position of both.

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Zusammenfassung Bei drei Arten von Stechmücken-Larven wurde die Schreck-Reaktion untersucht. Alle Larven verschwinden bei Reiz von der Wasseroberfläche, aber der Reaktions-Mechanismus unterscheidet sich bei den verschiedenen Arten. Aedes aegypti ist leichter als Wasser. Die Larven schwimmen aktiv zum Boden und kehren durch Aufwartstreiben passiv wieder zur Wasseroberfläche zurück. Die Larven von Culex und Anopheles sind schwerer als Wasser. Sie sinken bei Reiz passiv zum Boden, die Rückkehr zur Wasseroberfläche ist ein aktiver Prozess, welcher Schwimmbewegungen der Larven notwendig macht.Wiederholter Reiz führt zur Reizsättigung. Hat eine Larve aufgehört, auf einen Anreiz (z.B. Schwingung) zu reagieren, reagiert sie aber sofort auf einen anderen Reiz (z.B. Licht). Dieses deutet an, dass sich die Sinnes-Rezeptoren angepasst haben, dass aber die den Effekt bewirkenden Muskeln nicht ermüdet sind. Aedes-Larven sind nach Reiz negativ phototaktisch. Sie sind ausserdem positiv geotaktisch, aber die negative Phototaxis ist die stärkere Reaktion. Culex- und Anopheles-Larven erschlaffen, wenn sie gereizt werden, sie sinken in diesem Zustand passiv, sie reagieren nicht auf Wechsel in der Licht-Intensität.Die zu Aktivitätslosigkeit führende Temperatur ist um einige Grade kälter als die tiefste Temperatur, bei welcher Schreck-Reaktion noch stattfindet. Anpassung, welche vor 24 Stunden erfolgt, kann sowohl die zu Aktivitätslosigkeit führende Temperatur, als auch die Temperatur ändern, bei welcher noch eine Schreck-Reaktion ausgelöst wird.

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This work was started in the Entomology Department of the London School of Hygiene & Tropical Medicine, and was financed by the Medical Research Council.     

Production of the bioinsecticide Bacillus thuringiensis subsp. israelensis with deltamethrin increases toxicity towards mosquito larvae

Bacillus thuringiensis subsp. israelensis is a bioinsecticide used for larval mosquito control and it represents a safe alternative to chemical insecticides. Despite its environmental safety, it is less efficient and persistent than chemical insecticides. To bypass these limitations, we propose to combine the advantages of chemical and biological insecticides by producing Bti in a medium supplemented with a chemical insecticide (DDT, deltamethrin, permethrin, propoxur or temephos). Among the investigated insecticides, the addition of deltamethrin in the medium induced a higher toxicity (over 6·72‐fold) of the composite deltamethrin‐Bti towards mosquito larvae as compared to Bti alone. This was mainly due to the insertion of deltamethrin into the membranes of Bti spores, as evidenced by a quantification of membrane‐extracted deltamethrin by HPLC. This composite larvicide is a promising tool to decrease the quantity of chemicals dispersed in the environment, to increase the efficacy of Bti and to facilitate its widespread use as a transition between chemical and biological insecticides. Further experiments are required to characterize the mechanisms that underline the incorporation of deltamethrin into Bti to optimize the production and the toxicity of this composite larvicide.

Significance and Impact of the Study

This study is the first report of an increased efficacy of the mosquitocidal bioinsecticide Bacillus thuringiensis subsp. israelensis (Bti) when produced with a chemical insecticide. The results clearly demonstrate that deltamethrin is able to synergize the insecticidal activity of Bti through inclusion into spore membranes, reducing off‐target and nonspecific toxicity occurring when the chemical is used alone as sprays. This new composite chemical–biological insecticide can become an invaluable tool as an intermediate between single chemical usage and the widespread use of Bti, notably in developing countries with limited financial resources for intensive mosquito control campaigns.     

Transmission of viruses to mosquito larvae mediated by divalent cations

The two major groups of pathogenic viruses in mosquitoes are the occluded viruses, represented by baculoviruses and cypoviruses, and the non-occluded viruses, represented by the densoviruses and the iridoviruses. Baculoviruses, densoviruses, and iridoviruses are DNA viruses, while cypoviruses are the major group of RNA viruses reported from mosquitoes. Research on mosquito pathogenic viruses has been limited, in part, due to the inability to effectively transmit them to the larval mosquito host. Recently, there have been tremendous advancements in the ability to transmit mosquito baculoviruses and cypoviruses with the finding that transmission is mediated by divalent cations. Oral transmission of both baculoviruses and cypoviruses to mosquito larvae is enhanced by magnesium and inhibited by calcium ions. The current status of transmission for each of the major groups is reviewed with emphasis on the common role of divalent cations in transmission of the distantly related baculoviruses and cypoviruses.     

Preferential use of root litter compared to leaf litter by beech seedlings and soil microorganisms

Background and aims

Litter decomposition is regulated by e.g. substrate quality and environmental factors, particularly water availability. The partitioning of nutrients released from litter between vegetation and soil microorganisms may, therefore, be affected by changing climate. This study aimed to elucidate the impact of litter type and drought on the fate of litter-derived N in beech seedlings and soil microbes.

Methods

We quantified ¹⁵N recovery rates in plant and soil N pools by adding ¹⁵N-labelled leaf and/or root litter under controlled conditions.

Results

Root litter was favoured over leaf litter for N acquisition by beech seedlings and soil microorganisms. Drought reduced ¹⁵N recovery from litter in seedlings thereby affecting root N nutrition. ¹⁵N accumulated in seedlings in different sinks depending on litter type.

Conclusions

Root turnover appears to influence (a) N availability in the soil for plants and soil microbes and (b) N acquisition and retention despite a presumably extremely dynamic turnover of microbial biomass. Compared to soil microorganisms, beech seedlings represent a very minor short-term N sink, despite a potentially high N residence time. Furthermore, soil microbes constitute a significant N pool that can be released in the long term and, thus, may become available for N nutrition of plants.     

A novel bioassay system for evaluating the toxicity of Bacillus thuringiensis israelensis against mosquito larvae.

A bioassay system employing acutely toxic concentrations of a spore-crystal mixture of Bacillus thuringiensis subspecies israelensis against fourth instar larvae of Aedes aegypti (L.) is described. Individual larvae are separately exposed to toxin in glass-lined miniature wells or scintillation vials. This method is free from the deaths due to predation among larvae. Such larval deaths are commonly encountered in bioassay groups of 25 larvae as currently specified in the World Health Organization guidelines. Our method offers shortened testing time, increased accuracy, and improved statistical precision.