Tolypocladium cylindrosporum (Deuteromycotina: Moniliales), a fungal pathogen of the mosquito Aedes australis

The New Zealand strain of Tolypocladium cylindrosporum was cultured on Sabouraud dextrose agar medium under varying regimes of growth conditions. The isolate exhibited good tolerances to temperature (4–35 °C), pH (3–10) and salinity (0–7% NaCl). Optimal vegetative growth and sporulation were recorded between a temperature range of 20–30° C, pH of 5–6 and a salinity level of 0–2% NaCl. The North American isolate of the fungus showed similar tolerances, while the European isolate was less tolerant.     

Tolypocladium cylindrosporum (Deuteromycotina:Moniliales), a fungal pathogen of the mosquito Aedes australis

A laboratory fermenter was used to produce up to 12 l of infective Tolypocladium cylindrosporum blastoconidia in Sabouraud dextrose broth. Two media derived from coconuts were also demonstrated as suitable alternative systems for the production of viable blastoconidia. T. cylindrosporum conidia when dried at 37 degrees C and stored at 4 degrees C retained their viability for 10 months, but, when stored at 25 degrees C, the conidia lost viability after 2 months and blastoconidia did not survive the drying process. Distilled water suspensions were a simple, economic technique for the long-term storage of spores at both 4 and 25 degrees C. The adsorption of conidia onto silica gel crystals was a very suitable technique for the storage of stock culture material at 4 degrees C. The virulence, production and storage capabilities of both spore types were examined.     

Tolypocladium cylindrosporum (Deuteromycotina: Moniliales), a fungal pathogen of the mosquito Aedes australis

A blastoconidial preparation of the New Zealand isolate of T. cylindrosporum was tested in two field trials against larvae of the freshwater mosquito Aedes subalbirostris and the brackish water species Ae. australis. There was some reduction in the population of the Ae. subalbirostris larvae and larvae collected from the test pools exhibited signs of fungal infection when reared in the laboratory. In the brackish pools there was no observable decrease in the larval populations even though Ae. australis is the natural host of T. cylindrosporum [NZ]. Water volume fluctuations in the test pools were a major difficulty experienced when monitoring the results.Tests against field-collected non-target fauna indicated that T. cylindrosporum is non-specific in action. Species of daphnid, dixid larvae and copepods were all susceptible to infection at varying degrees.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

Effects of Tolypocladium cylindrosporum and its secondary metabolites, efrapeptins, on the immune system of Galleria mellonella larvae

Interactions between Tolypocladium cylindrosporum (Deuteromycetes), its metabolite, efrapeptins, and the insect immune defense were investigated in vivo and in vitro. In the different phagocytosis studies, Bacillus cereus spores which had been labelled with fluorescein-isothiocyanate (FITC) were used. In vitro studies showed that efrapeptins inhibit phagocytic activity of Galleria mellonella (Lepidoptera: Pyralidae) haemocytes. The response was dose-related. Efrapeptins significantly reduced the number of nodules formed in response to an injection of zymosan supernatant. Phenoloxidase (PO) activation system contained in haemocyte lysate (HLS) was not affected by efrapeptins. In vivo studies when larvae were injected with efrapeptins also revealed that efrapeptins did not affect PO activities and total haemocyte count (THC) after 1 and 6 h post-injection. However, 12 h post-injection there was a significant inhibition of PO activities in HLS. There was also no significant reduction of PO activities and THC when larvae were injected with Tolypocladium cylindrosporum spores until 24 h post-injection. However, PO activities were suppressed and THC reduced 48 h post-treatment of larvae with spores. This study suggests that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes.     

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.     

Utility of six fungicides for selective isolation of Evlachovaea sp. and Tolypocladium cylindrosporum

The effects of the fungicides dodine, benomyl, thiabendazole, mancozeb, cupric sulfate, and copper oxychloride were examined in vitro upon germination and further development of Evlachovaea sp. and Tolypocladium cylindrosporum. Fungicidal activity depended on concentrations and varied among products, fungi and the strains tested. Depending on the fungicidal concentration, germination of conidia was induced but germlings produced neither mycelium nor new conidia. There was a good recovery of both Evlachovaea sp. and T. cylindrosporum from previously sterilized soils with fungicide-supplemented medium. Fungi were resistant to copper oxychloride up to 30 g/l, and this fungicide was found to have no utility for a selective medium. Minimal fungicide concentrations for successful isolations were 1 mg/l for benomyl, 200 mg/l for cupric sulfate, 50 mg/l for dodine, 100 mg/l for mancozeb, and 4 mg/l for thiabendazole. Thiabendazole, which is easy to obtain and can be used in low quantities, showed the greatest utility for a selective medium with these entomopathogenic fungi. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

高山弯颈霉(Tolypocladium nubicola),中国弯颈霉属一新记录种

报道了分离自土壤的一个弯颈霉属新记录种 ,即高山弯颈霉 (TolypocladiumnubicolaBissett)。其主要鉴别特征是 :①在查氏培养基上 ,2 5℃ ,14d ,直径达 30～ 35mm ,平展 ;②瓶梗直接着生于菌丝或简单的分生孢子梗上 ,有再育现象 ,基部拟椭圆形膨大或球形膨大 ,(2 .6～ 7.2 ) μm× (1.3～ 3.2 ) μm ,颈部细长 ,常弯曲 ,(3.0～6 .3) μm×0 .3μm ;③分生孢子单生 ,柱状至椭圆形 ,(1.6～ 3.8) μm× (0 .8～ 1.2 ) μm ,外无黏液层。     

Taxonomic and phylogenetic analysis of the Oomycota mosquito larvae pathogen Crypticola clavulifera

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.     

Mycovirus effect on the endophytic establishment of the entomopathogenic fungus Tolypocladium cylindrosporum in tomato and bean plants

Two endophytic strains of the entomopathogenic fungus Tolypocladium cylindrosporum, originally isolated from the grass Festuca rubra, were artificially inoculated in tomato and bean plants. Strains 11-1L and 11-0BR were isolated from asymptomatic leaf fragments of both plant species at 3, 7, 14, 21, and 35 days after their inoculation. The percentage of leaf fragments infected by the fungus in inoculated leaves decreased at each sampling time, and no systemic colonization of the plants occurred. The two T. cylindrosporum strains tested were isogenic, differing in the infection by the victorivirus TcV1, harboured by strain 11-1L, but not by 11-0BR. The percentage of infected leaf fragments in leaves inoculated with the virus infected strain was greater in bean than in tomato plants, while the virus-free strain was more successful in tomato than in bean plants. This result suggests that the mycovirus infection can affect the adaptation of T. cylindrosporum to particular host plants.     

Establishment of a mosquito cell line fromHaemagogus equinus larvae

Summary The establishment and characterization of aHaemagogus equinus mosquito cell line (GML-HE-12) are described. The cells are diploid (2N=6) and seem to be free of contaminants. Their susceptibility to 13 arboviruses was tested. Eleven of the viruses multiplied in this cell line; six of these viruses still showed titers of 4 log₁₀ plaque forming unit/ml or greater at 33 d postinoculation. No overt cytopathologic effect was observed.     

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.

---

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.

---

---

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

Beauveria tenella as a control agent for mosquito larvae

Beauveria tenella was found to be a naturally occurring pathogen in field populations of Aedes sierrensis larvae. Bioassays showed that B. tenella is also pathogenic for larvae of A. aegypti, A. dorsalis, A. hexodontus, Culex pipiens, C. tarsalis, and Culiseta incidens. Inoculation of 5 × 10³ or 5 × 10⁵ blastospores per milliliter into breeding sites of A. sierrensis produced marked reductions in the number of emerging adults.     

A new serotype of Bacillus thuringiensis from Colombia toxic to mosquito larvae.

During a survey conducted in Colombia a new isolate of Bacillus thuringiensis that showed toxicity toward Culex quinquefasciatus, Cx. pipiens, Aedes aegypti, and Anopheles stephensi larvae was isolated. Parasporal crystals were spherical in shape and showed a great degree of similarity with those produced by the reference strain of Bacillus thuringiensis subsp. israelensis. Supernatant fraction of the whole culture was not toxic, and heat-stable exotoxin production was negative. Catalase, urease, arginine dihydrolase, amylase, lecithinase, acetyl-methyl-carbinol, and gelatinase production were positive. Hemolysis on sheep blood agar was alpha-type. The isolate 163-131 showed natural resistance to azolocillin and was sensible to cephoperazone, cephalotin, nalidixic acid, and trimetoprin sulfametoxazole. Flagellar agglutination showed a specific H 30 antigen which allows individualization of this strain as a new serotype and the subspecies name of medellin is proposed.