Dependence of the entomopathogenic fungus,Beauveria bassiana,on high humidity for infection of Rhodnius prolixus

Mycopathologia - The impact of relative humidity (RH) on the infective potential of the isolate Bb INRA 297 of Beauveriabassiana (Bals.) Vuillemin (Deuteromycotina: Hyphomycetes) against first in...     

Temperature and moisture requirements for conidial germination of an isolate of Beauveria bassiana,pathogenic to Rhodnius prolixus

The effects of temperature, relative humidity and water activity on germination of conidia of an isolate of Beauveria bassiana (Bals.) Vuill. pathogenic to the triatomine vector of Chagas' disease, Rhodnius prolixus Stål., were investigated in vitro. Germination occurred at temperatures between 15 °C and 35 °C under saturated atmosphere and the optima ranged from 25 °C to 3O °C. At the extreme temperatures tested (15 °C and 35 °C) the germination process was delayed, but germination rates reached more than 95%. Germination of B. bassiana conidia was strongly affected by moisture conditions. The availability of water, in both atmospheric and liquid conditions, caused changes in germination times as well as in germination rates. For example, at 25 °C + O.5 °C, germination took place within 20 h at 95.5% RH, whereas it needed 72 h of incubation at 90% RH. Germination times increased as the water activity declined from 0.96 a_w to 0.92 a_w. Below 0.92 a_w, o germination was observed after a 72 h incubation time.     

Effects of Fluctuating Moisture and Temperature Regimes on Sporulation of Beauveria bassiana on Cadavers of Rhodnius prolixus

The effects of both moisture and temperature on the sporulation of an isolate of Beauveria bassiana , highly virulent to the Chagas' disease vector, Rhodnius prolixus , were studied in the laboratory under fluctuating regimes. Quantitative assays for conidial production from funguskilled cadavers from 10-day-old tests showed that recycling was highly dependent on the duration of exposure to high relative humidity (RH). On first-instar larval cadavers, sporulation reached more than 10 5 conidia/nymph under most regimes with a favourable phase combining high humidity (97% RH) and temperatures of 20 or 25 C for at least 12 h/day. Sporulation on third- and fifth-instar nymphs required a longer period (16 h/day or more) of favourable conditions (97% RH and 25 C) per day. The intensity of the conidial production from Rhodnius cadavers was enhanced when diurnal temperatures were high (28 and 35 C). From a vector control standpoint, daily high humidity requirements appear to be a crucial constraint. The recycling ability of B. bassiana on fungus-killed R. prolixus is most likely to contribute to the regulation of this triatomine vector if applications are made during the rainy seasons and in the most favourable habitats.     

Relative susceptibility of different stages of Rhodnius prolixus to the entomopathogenic hyphomycete Beauveria bassiana.

Laboratory bioassays were conducted to determine the relative susceptibility of eggs, 1st-, 3rd-, 5th-instar nymphs and adults of Rhodnius prolixus to one isolate of the entomopathogenic hyphomycete, Beauveria bassiana. Treatments consisted of directly spraying on insects of increasing doses of inoculum (3 x 10(2) to 3 x 10(5) conidia per cm2). Mortality due to all doses of conidia was very high in the five tested stages of the target insect. Experiments on eggs demonstrated that the fungal isolate was able to kill eggs before they hatched. Both time-mortality and dose-mortality responses showed that the susceptibility of R. prolixus varied according to its stage of development and increased with age. As a matter of fact, at the dose of 3 x 10(3) conidia per cm2, LD 50 varied between 11.2 days in 1st-instar nymphs and 6.4 days in both 5th-instar nymphs and adults. Comparison of LD50 permitted to estimate that 1st-instar nymphs were about 700-fold less susceptible than the two oldest stages.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Influence of starvation and blood meal-induced moult on the susceptibility of nymphs of Rhodnius prolixus Stål (Hem., Triatominae) to Beauveria bassiana (Bals.) Vuill. infection

Abstract: The influence of starvation and nutritional status on the time–mortality response of nymphs of Rhodnius prolixus Stål (Hem., Reduviidae) contaminated with Beauveria bassiana (Bals.) Vuillemin (Deuteromycotina, Hyphomycetes) was determined. Because of the induction of the moulting process by the blood meal, first-instar nymphs were less susceptible on day 12, when they were in pre-moult. Starvation did not affect nymphal susceptibility to B. bassiana up to a period of 1 month. There was no significant difference in time–mortality responses between unfed nymphs and blood-engorged nymphs. This information is discussed for microbial control of vectors with respect to the importance of periods of starvation in triatomines under natural conditions.     

Enhanced efficacy of Beauveria bassiana for red flour beetle with reduced moisture

Red flour beetle, Tribolium castaneum (Herbst), is a major pest of stored and processed grains that is tolerant of Beauveria bassiana (Balsamo) Vuillemin under most conditions. Laboratory assays demonstrated that desiccating conditions improved the efficacy of the fungus. For T. castaneum larvae exposed to B. bassiana at different vapor pressure deficits (VPDs), the LC50 potency ratios were 2.3 for 2.42 kPa and 3.9 for 3.31 kPa compared with 1.06 kPa. There were significant effects of moisture on B. bassiana-associated mortality even with brief exposure to low VPD before or at the beginning of exposure to fungus. When T. castaneum larvae were held at 3.31 kPa 1 d before exposure to B. bassiana and/or in the first 1 or 2 d of exposure to the fungus, there was significantly greater mortality for all of the low moisture treatments than occurred with continuous incubation at 1.06 kPa. B. bassiana efficacy for adult T. castaneum was significantly better for VPDs of 3.56 or 3.78 than 1.06 kPa. Weight gain over 4 d of incubation with moisture held constant was significantly less at a VDP of 3.31 kPa than at 1.87 or 1.06 kPa. Treatment with fungus did not affect weight gain significantly. There was no significant difference in larval water content among treatments.     

Beauveria bassiana infection of eggs of stored-product beetles

Beauveria bassiana (Balsamo) Vuillemin was tested under maximum challenge conditions with an estimated dose of 1.1 × 10⁵ conidia/mm² for its effects on eggs of four of the major beetle pests of stored grain and grain products. When ambient relative humidity (RH) was 92%, hatch of fungus-treated Rhyzopertha dominica (Fabricius) eggs was 13% versus 58% for control eggs, and hatch of treated Tribolium castaneum (Herbst) was 17% versus 51% for controls. There was no fungus effect at RH of 48 and 73%. Fungus treatment of Cryptolestes ferrugineus (Stephens) and Oryzaephilus surinamensis (Linnaeus) eggs had no effect. Sectioned T. castaneum eggs demonstrated that the fungus penetrates and infects them.     

锌离子响应转录激活因子ZafA对球孢白僵菌锌离子利用及生防潜能的影响

球孢白僵菌作为丝孢类昆虫病原真菌,已广泛应用于农林害虫的生物防治,但是田间多变的环境影响了真菌制剂的效能。此外,真菌侵染宿主后,宿主体内的环境也影响真菌的增殖和侵染速度。为研究球孢白僵菌对环境中酸碱度及微量元素的平衡能力,本研究初步探讨了锌离子响应转录激活因子ZafA与真菌生长繁殖、抗逆能力、毒力以及锌离子利用的关系。结果表明,敲除zafA削弱了真菌生长繁殖和孢子萌发的能力,增加了菌株对氧化、高渗、孢壁干扰剂以及紫外胁迫的敏感性,杀虫毒力显著下降,并抑制了相关性状基因的表达水平。基因敲除菌株无法在锌离子缺失的条件下生长,且zafA基因和锌离子转运蛋白编码基因zrf1–8的表达水平会受到酸碱度以及锌离子浓度的影响。由此可见,ZafA不仅直接影响球孢白僵菌利用锌离子的能力,还与球孢白僵菌抗逆能力和毒力密切相关,本研究为提高生防真菌环境适应性和发挥毒力提供新的依据。     

Thermosensation and the TRPV channel in Rhodnius prolixus

The thermal sense of triatomine bugs, vectors of Chagas disease, is unique among insects. Not only do these bugs exhibit the highest sensitivity to heat known in any animal up to date, but they can also perceive the infrared radiation emitted by the body of their warm-blooded hosts. The sensory basis of this capacity has just started to be unravelled. To shed additional light on our understanding of thermosensation, we initiated an analysis of the genetic basis of the thermal sense in Rhodnius prolixus. We tested the hypothesis that a TRPV (transient receptor potential vanilloid) channel receptor is involved in the evaluation of heat in this species. Two different approaches were adopted. Initially, we analysed the expression of a TRPV candidate for this function, i.e., RproIav, in different tissues. Subsequently, we tested the effects of capsaicin and capsazepine, two molecules known to interact with mammal TRPV1, using three different behavioural protocols for evaluating thermal responses: (1) proboscis extension response (PER), (2) thermopreference in a temperature gradient and (3) spatial learning in an operant conditioning context. Bioinformatic analyses confirmed that the characteristic features typical of the TRPV channel subfamily are found in the RproIav protein sequence. Molecular analysis showed that RproIav is expressed in R. prolixus, not only in the antennae, but also in other body structures bearing sensory organs. Behavioural experiments consistently revealed that capsaicin treated insects are less responsive to heat stimuli and prefer lower temperatures than non-treated insects, and that they fail to orient in space. Conversely, capsazepine induces the opposite behaviours. The latter data suggest that triatomine thermoreception is based on the activation of a TRP channel, with a similar mechanism to that described for mammal TRPV1. The expression of RproIav in diverse sensory structures suggests that this receptor channel is potentially involved in bug thermoreception. This constitutes solid evidence that thermosensation could be based on the activation of TRP receptors that are expressed in different tissues in R. prolixus. Whether RproIav channel is a potential target for the compounds tested and whether it mediates the observed effects on behaviour still deserves to be confirmed by further research.     

A preliminary evaluation of the potential of Beauveria bassiana for bed bug control

Residual biopesticide treatments of Beauveria bassiana were tested against the bed bug Cimex lectularius. An oil formulation of conidia was applied to different substrates. Bed bugs were exposed for 1h, transferred to an unsprayed environment and monitored for mortality. Separate bioassays evaluated the effect of bed bug strain, sex, life stage, and exposure substrate on mortality. Rapid mortality was observed in all bioassays, with bed bugs exposed to treated jersey knit cotton dying most rapidly. A further assay demonstrated efficient autodissemination of conidia from exposed bed bugs to unexposed bed bugs within artificial harborages.     

Genotype-environment interactions for insect growth in constant and fluctuating temperature regimes

Experiments on life history genetics are usually performed using constant temperature environments in the laboratory. However, the dynamics of insect growth can be influenced profoundly by daily fluctuations in temperature such as those which characterize field environments. We report here on experiments using different stocks and selected lines of a tropical butterfly, Bicyclus anynana, to examine whether genotype-environment interactions occur for three traits describing pre-adult growth. These traits were measured over two pairs of environments differing in mean temperature, each of which had a constant, and a cycling temperature regime. Development time, pupal weight and growth rate show genotype-environment interactions, especially at comparatively low average temperatures. Researchers should, therefore, take care when extrapolating from the form of genetic covariance matrices and ''trade-offs'' among life history traits found in constant temperature environments to those likely to occur in nature. <br>     

Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii

The commercial use of entomopathogenic fungi and their products as mycoinsecticides necessitates their registration. Worldwide, several registration guidelines are available, however, most of them focus on similar or even the same safety issues. With respect to the two entomopathogenic fungi, Beauveria bassiana (Bals.-Criv.) Vuill. and Beauveria brongniartii (Sacc.) Petch, many commercial products have been developed, and numerous papers on different biological, environmental, toxicological and other safety aspects have been published during the past 30-40 years. The aim of the present review is to summarise these data. The following safety issues are presented: (1) identity of Beauveria spp.; (2) biological properties of Beauveria spp. (history, natural occurrence and geographical distribution, host range, mode of action, production of metabolites/toxins, effect of environmental factors); (3) analytical methods to determine and quantify residues; (4) fate and behaviour in the environment (mobility and persistence in air, water and soil); (5) effects on non-target organisms (non-target microorganisms, plants, soil organisms, aquatic organisms, predators, parasitoids, honey bees, earth worms and nontarget arthropods); (6) effects on vertebrates (fish, amphibia, reptiles and birds); and (7) effects on mammals and human health. Based on the present knowledge it is concluded that both Beauveria species are considered to be safe.     

Trypanosoma cruzi: effects of infection on cathepsin D activity in the midgut of Rhodnius prolixus

Cathepsin D activity was estimated in midgut homogenates from Rhodnius prolixus, uninfected and experimentally infected with Trypanosoma cruzi, at different times after blood ingestion. No enzyme activity was found in the anterior midgut and rectum. In the posterior midgut, enzyme activity was found both in lumen and wall. In starved uninfected insects, in lumen and wall, cathepsin D activity was high, decreasing to a constant rate at 1-15 days after feeding. In insects infected with T. cruzi cathepsin D activity increased 1 and 3 days after blood meal. We suggest that these changes in cathepsin D activity in R. prolixus posterior midgut are due to the establishment of T. cruzi infection.     

Effects of gamma irradiation on the development of Trypanosoma rangeli in the vector Rhodnius prolixus

Studies on the effects of gamma radiation on the infectivity of Trypanosoma rangeli (strain H14) for the vector Rhodnius prolixus revealed that (i) the LD(50) (lethal dose for 50% of bugs) for uninfected insects was 4147 rads; (ii) irradiated insects with a dose of 1200 rads subsequently infected with the flagellates exhibited a mortality of 45%, while uninfected irradiated insects showed a mortality of 5%, and infected nonirradiated insects exhibited 10% mortality; (iii) flagellates were present in the hemolymph of irradiated insects 7 days postinfection (p.i.), while in nonirradiated insects the parasites appeared in the hemocoel 18 days p.i.; (iv) T. rangeli infection decreased the number of hemocytes significantly and induced the formation of nodules in the hemolymph of both irradiated and nonirradiated insects; and (v) gamma irradiation affected the ultrastructural organization of the epithelial cells of the small intestine, principally the perimicrovillar membranes and microvilli. In this paper, we discuss the significance of the intestinal microenvironment of R. prolixus with regard to its interaction with T. rangeli.     

Embryonic development of the hemipteran insect Rhodnius prolixus

The embryonic development of the hemipteran insect Rhodnius prolixus was studied by use of contemporary light and electron microscopy. Embryos were staged according to days postoviposition. Eggs laid on day one complete blastoderm formation and anatrepsis, the first phase of blastokinesis, by day 5. The embryo develops in a cephalocaudal orientation which is 180° to the anteroposterior axis of the egg. Subsequent development, prior to the second phase of blastokinesis (katatrepsis), leads to segmentation of the germ band, evagination of appendages, and histogenesis of germ layers. Concomitantly with these events, the amnion undergoes dramatic change. By day 7 the embryo begins a 180° revolution while migrating to the ventral surface of the yolk. This restores its polarity with respect to that of the egg and facilitates hatching. The serosa contracts, pulling the amnion and embryo anteriorly. Eventually the serosa is internalized at a point dorsal to the head and the lateral walls of the embryo grow up and surround the yolk. Development continues until day 15 when the embryo hatches as a first instar larva.     

Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.