Acid phosphatases in the haemolymph of the desert locust, Schistocerca gregaria, infected with the entomopathogenic fungus Metarhizium anisopliae

A comparison has been made between the effects of wounding, chemical stimulation of the immune system and fungal infection on acid phosphatase (AcP) activity in the haemolymph of the desert locust, Schistocerca gregaria. Untreated control locusts had constitutive levels of AcP. As a lysosomal enzyme, AcP may have a role in autophagy and cell turn over as well as defence. Injection of saline and beta-1,3-glucan caused significant increases in haemocyte and plasma AcP. AcP activity also increased in the haemolymph on the 3rd day after inoculation with the entomopathogenic fungus M. anisopliae var acridum. This coincided with a decline in the total haemocyte count and a marked reduction in the proportion of plasmatocytes and coagulocytes that stained positive for AcP. Therefore a priori it seemed unlikely that the extra AcP in infected insects came from the host. A fungal origin for the enzyme was suggested by the identification of AcP isoforms from haemolymph of different treatments. Control inoculated (oil only) insects had an AcP at a pI of 4.3 that was stimulated further by the injection of laminarin. Additional isoforms appeared at around 7.3-7.5 in the laminarin treatment. However, the 4.3 isoform appeared to be suppressed in the insects infected with M. anisopliae var acridum. The band intensity was more like that of the control than the laminarin-injected insects. Two new isoforms appeared later on in infection. These enzymes had pIs that corresponded to some of the AcPs produced in vitro by the fungus. The results are discussed in the light of the possible benefits of secreted fungal acid phosphatases to the pathogen.     

The effect of Metarhizium anisopliae var acridum on haemolymph energy reserves and flight capability in the desert locust, Schistocerca gregaria

Adult desert locusts, Schistocerca gregaria , 3 days after inoculation with the entomopathogenic fungus Metarhizium anisopliae var acridum , had significantly less carbohydrate and lipid in the haemolymph than controls. This was not due to reduced food intake as 3 days of complete starvation had no effect on haemolymph titres of energy reserves in controls. Furthermore injection of an extract of the corpora cardiaca (the source of adipokinetic hormone, AKH) caused a large significant increase in haemolymph lipid in mycosed locusts, indicating the availability of significant quantities of lipid in the fat body, the target for AKH. Haemolymph carbohydrate declined significantly during tethered flight of control locusts but not in mycosed individuals. An injected supplement of trehalose significantly boosted flight performance of mycosed insects but not controls. The results are discussed in the light of the hypothesis that the poor flight capability of mycosed locusts is due in part to a fungus-induced reduction in mobile energy reserves.     

Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta migratoria infected with Metarhizium anisopliae strain CQMa102

Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa 102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis.     

Inhibition of fungal growth in thermoregulating locusts,Locusta migratoria,infected by the fungus Metarhizium anisopliae var acridum

The locust, Locusta migratoria, has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum. We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.     

黄绿绿僵菌对褐飞虱和白背飞虱不同发育阶段的病原性的研究

本文报道了不同孢子浓度下黄绿绿僵菌对褐飞虱和白背飞虱不同发育阶段的易感性和毒力的研究。实验设10.5孢子/mm2,116.7孢子/mm2和1027.1孢子/mm2三种孢子剂量,两种飞虱分为幼龄若虫(1龄和2龄若虫)、高龄若虫(3、4、5龄若虫)和成虫三个发育阶段。实验发现褐飞虱与白背飞虱的三个发育阶段对黄绿绿僵菌的不同浓度的孢子液有不同程度的易感性。黄绿绿僵菌对褐飞虱幼龄若虫的毒力指标LT50在三种孢子剂量下依次为>21、20.82和16.55;对高龄若虫的LT50在三种孢子剂量下依次为17.68、15.49和13.98;而对成虫的LT50在三种孢子剂量下依次为17.10、12.57和9.14。黄绿绿僵菌对白背飞虱幼龄若虫的毒力指标LT50在三种孢子剂量下依次为>21、17.29和13.13;对高龄若虫的LT50在三种孢子剂量下依次为16.94、15.02和13.03;而对白背飞虱成虫的LT50在三种孢子剂量下依次为12.78、10.16和7.64。二者的成虫的易感性比若虫的易感性强,高龄若虫的易感性比幼龄若虫的强。白背飞虱比褐飞虱对黄绿绿僵菌更加敏感。二者的死亡率随孢子浓度的增大而增大。     

PATHOGENICITY OF METARHIZIUM ANISOPLIAE VAR. ACRIDU TO THE DEVEOLPMENTAL STAGES OF BROWN PLANTHOPPER NILAPARVATA LUGENS STÅL AND SOGATELLA FURCIFERA (HORVATH)

Abstract  The susceptibility and virulence of entomopathogenic fungus Metarhizium anisopliae var. acridum to the stages of brown planthopper (BPH), Nilaparvata lugens (Stål) and whitebacked planthopper (WBPH), Sogatella furcifera (Horvath) were investigated under laboratory conditions. Three dosages of M. anisopliae var. acridum ranging from 10.5, 116.3 and 1027.1 conidial/mm² were used in the experiment. The tested stages of host included three developmental stages, young nymphs (1–2 instars), old nymphs (3–5 instars) and adults. It was found that all tested stages of the planthoppers were susceptible to the fungal infection. The degree of virulence LT₅₀ of M. anisopliae var. acridum against young nymphs of N. lugens are >21, 20.82 and 16.55, respectively with the 3 dosages, the LT₅₀ of the fungus against the old nymphs are 17.68, 15.49 and 13.98, respectively with the 3 dosages; the LT₅₀ of the fungus against the adults are 17.10, 12.57 and 9.14 respectively with the 3 dosages. The degree of virulence LT₅₀ of M. anisopliae var. acridum on young nymphs of S. furcifera are >21, 17.29 and 13.13, respectively with the 3 dosages _; the LT₅₀ of the fungus against the old nymphs are 16.94, 15.02 and 13.03, respectively with the 3 dosages; the LT₅₀ of the fungus against the adults are 12.78, 10.16 and 7.64, respectively with the 3 dosages. Adults were more susceptible to M. anisopliae var. acridum infection than their nymphs and the young nymphs were most resistant to the fungal infection. The cumulative mortality of each stage was dosage-dependent. Of all the developmental stages, WBPH was more susceptible than BPH to M. anisopliae var. acridum infection with the same dosages.     

Acid phosphatases of Metarhizium anisopliae during infection of the tobacco hornworm Manduca sexta

Three acid phosphatase (AcP) isozymes, pI 8.1, 8.0 and 7.8, were isolated, purified and partially characterised from optimised cultures of the entomopathogenic fungus Metarhizium anisopliae. The enzymes had similar molecular masses (approximately 44.0 kDa), and could degrade sugar phosphates found in the haemolymph of a host insect, the tobacco hornworm Manduca sexta. The AcP activity in haemolymph of mycosed insects increased significantly over controls, and some new isozymes were present. The infection-related isoforms were similar in molecular mass and pI to some of the in vitro AcP isozymes of M. anisopliae. Results of dot blot and Western blot analyses using anti-AcP antibodies suggested that at least one Metarhizium phosphatase isoform was present in haemolymph of infected caterpillars. Antibodies did not cross-react with immune (chemically stimulated) or non-immune haemolymph from Manduca sexta. Consistent with the appearance of highly active fungal phosphatase in caterpillar blood, free phosphate concentration increased dramatically during the late stages of infection to a level two to five times that of controls. Phosphate was limiting to growth of the fungus at the concentration found in control haemolymph and supplementation of phosphate significantly increased fungal growth in vitro in haemolymph. These results suggest that Metarhizium AcP may play a key role in providing phosphorus for fungal growth at the expense of the insect.     

Effet de l'application d'un mélange lambda-cyhalothrin (pesticide chimique) et de spores de Metarhizium anisopliae (flavoviride) var. acridum Driver and Milner (biopesticide) appliqué sur les larves de sauteriaux au Mali

A mixture of lambda-cyhalothrin (lambda-cyhalothrin: chemical insecticide) and Metarhizium anisopliae ( flavoviride ) var. acridum Driver and Milner, an entomopathogenic fungus (bioinsecticide) was used for grasshopper control in Mali. An oil-based formulation of Metarhizium anisopliae ( flavoviride ) var. acridum Driver and Milner has been developed by LUBILOSA a collaborative project for locust and grasshoppers control. It takes 6 to 10 days for the biopesticide to kill the hosts, which is not a problem for larvae in fallows because they will die before reaching the farmers' fields. However, if crops are infested by adults, the farmers can not wait for 6 to 10 days. An experiment was conducted in Mali using a mixture of a biopesticide and chemical pesticide. The mixture of lambda-cyhalothrin (chemical insecticide) and Metarhizium anisopliae ( flavoviride ) var. acridum (biopesticide: oil-based entomopathogenic fungus spore suspension) was applied to nymphs of Sahelian grasshoppers, using ultra low volume (ULV) sprayers. Both the mixture and lambda-cyhalothrin alone gave quick mortality, with slightly higher mortality for the mixture. Mortality due to the Metarhizium treatments began 2 days after application and subsequently reached similar levels of mortality to the lambda-cyhalothrin mixture treatments. The efficacy of the mixture was greater than Metarhizium alone. The efficacy of lambda-cyhalothrin reached 80% on the day following application, but declined after 10 days, due probably to immigration of untreated grasshoppers.     

Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum

AIMS: To examine the ability of Agrobacterium to attach to Metarhizium anisopliae var. acridum strain CG423 under co-cultivation and to develop an Agrobacterium-mediated method of gene delivery into strain CG423, a promising agent for biological control of grasshoppers. METHODS AND RESULTS: The co-cultivation of Agrobacterium tumefaciens and M. anisopliae var. acridum was analysed under scanning electron microscopy. We observed that Agrobacterium attached to and formed aggregates around Metarhizium conidia and germ tubes. We also observed the occurrence of fibril-like structures connecting neighbouring bacterial-fungal cells. The Agrobacterium-mediated transformation was applied using two binary vectors carrying a benomyl resistance gene as a selection marker. The efficiency of transformation was up to 53 transformants per 10(5) target conidia. High mitotic stability of the transformants (89-97%) was demonstrated after five successive transfers on non-selective media. Molecular analysis revealed the occurrence of high frequency of gene conversion. CONCLUSIONS: In our study, we report that A. tumefaciens strain AGL-1 attaches to and genetically transforms the entomopathogenic fungus Metarhizium anisopliae var. acridum. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time, the attachment of Agrobacterium to fungal cells opening new avenues for the study of this essential step of the T-DNA transfer process. Considering the efficiency of the transformation protocol herein described, this is a useful tool for gene disruption in M. anisopliae var. acridum.     

Adult survival, maturation, and reproduction of the desert locust Schistocerca gregaria infected with the fungus Metarhizium anisopliae var acridum

Studies were conducted with two different doses of Metarhizium anisopliae var acridum to examine the effects on survival and reproductive potential of adult Schistocerca gregaria under conditions that either limited thermoregulation or enabled optimal thermoregulation. Adult S. gregaria infected with the fungal pathogen showed either a rapid and high mortality at relatively constant temperatures or a much reduced mortality and lengthened survival time when allowed to thermoregulate. Mortality rate varied from >90% after 10 days under constant temperature conditions to 66% after 70 days under optimal thermoregulatory conditions. Effects of infection on maturation and reproduction depended on the age of the adults at the time of inoculation, the nighttime temperature regime, the fungal dose, and the length of time of the monitoring period. No difference in reproductive behaviors in treated and control insects were found in one experiment that utilized older adults and was conducted over 25 days. In a second experiment with newly fledged locusts, differences in maturation rates and total reproductive output were observed due to infection. The results from these experiments are discussed in terms of the potential of M. anisopliae var acridum to alter the balance of insect endocrine systems and the importance of the assessment of behavioral changes and their impact on microbial control agents in the long term.     

人趋化因子MIP-3α的原核可溶性表达及趋化活性分析

目的:克隆人趋化因子MIP3α,进行原核表达并初步鉴定其趋化活性。方法:从人扁桃体中提取总RNA,进行RTPCR,扩增MIP3α成熟蛋白基因,重组于pET32a(+)载体,转化大肠杆菌BL21TrxB(DE3),进行融合表达,Westernblot验证融合蛋白,金属离子亲和层析,肠激酶酶切,弱阳离子交换层析,得到纯化的MIP3α蛋白,趋化试验鉴定其趋化活性。结果:成功构建了MIP3α天然蛋白的硫氧还蛋白融合表达载体,表达并纯化出MIP3α蛋白,Westernblot证明融合蛋白能与羊抗人MIP3α抗体结合,纯化的MIP3α蛋白能趋化HEK293CCR6稳定转染细胞。结论:构建的天然MIP3α融合表达载体以可溶性蛋白的方式稳定表达MIP3α,初步纯化得到的MIP3α具有趋化HEK293CCR6稳定转染细胞的活性。     

Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two pathogens of the desert locust, Schistocerca gregaria under laboratory conditions

The interaction between two pathogens, the microsporidian Paranosema locustae Canning and the fungus Metarhizium anisopliae var. acridum Driver and Milner was studied under laboratory conditions in an attempt to develop an improved method of microbial control for the desert locust, Schistocerca gregaria Forsk?l. Fifth-instar locust nymphs, reared in the laboratory, were treated with various concentrations of one of the two pathogens or with both pathogens. The numbers of locusts killed were recorded each day and the production of pathogen spores within the dead locusts was assessed at the end (day 21) of each experiment. Locust nymphs treated with both P. locustae and M. anisopliae died sooner than nymphs infected with only one of the pathogens. At the lower concentrations of pathogen tested, the effects of the two pathogens were additive. At the higher concentrations the combined effects were synergistic. In terms of locust mortality, there was no evidence of any antagonistic effects between the two pathogens. However, the production of spores by P. locustae was reduced considerably when the host insects were infected also with M. anisopliae. For example, nymphs treated initially with P. locustae and then treated 3 and 10 days later with M. anisopliae produced 3-20 times and 2.5-8 times fewer spores, respectively, than nymphs treated only with P. locustae. Hence, in areas where M. anisopliae is applied, the natural persistence of P. locustae in the local grasshopper and locust populations may be diminished.     

Fungal infection causes changes in the number,morphology and spreading ability of Galleria mellonella haemocytes

The most effective and important strategy in the insect immune response is based on cellular reactions incorporating haemocytes. The present study uses Galleria mellonella (Lepidoptera: Pyralidae) as a host to study the pathogenesis caused by the entomopthoralean fungus Conidiobolus coronatus (Entomophthorales). Five types of haemocytes with different morphologies and behaviour are observed in the haemolymph of G. mellonella: granulocytes (GRs), plasmatocytes (PLs), spherulocytes (SPs), oenocytes (OEs) and prohaemocytes (PRs). During in vitro cultivation, three morphological subtypes of PLs are distinguished: flattened PLs, sun‐like PLs and oval PLs. In fresh smears of haemolymph observed under phase‐contrast microscopy, only flattened PLs are identified. No morphological changes are observed between fresh smears and in vitro cultures for GR, OE, SP and PR. Haemocytes cultured in vitro form a cellular network composed of PLs and GRs. Changes in the numbers, morphology and behaviour of haemocytes induced by fungal infection are compared with those observed in normally‐developing untreated larvae. Infection results in a significant drop in the number of haemocyte types. Fresh smears of haemocytes from mycosed larvae reveal malformed OEs, vacuolized PLs and GRs, as well as PLs with apoptotic blebs. Haemocytes from mycosed larvae incubated in vitro look similar, with degranulated GRs and vacuolized PLs forming microaggregations, as well as deformed OEs; only the SPs remain unharmed. Fungal infection impairs the ability of haemocytes to attach and spread on the culture dish. The actin cytoskeleton of haemocytes from mycosed larvae appear disorganized.     

Identification of an extracellular acid trehalase and its gene involved in fungal pathogenesis of Metarizium anisopliae

Trehalose is the main sugar in the haemolymph of insects and is a key nutrient source for an insect pathogenic fungus. Secretion of trehalose-hydrolysing enzymes may be a prerequisite for successful exploitation of this resource by the pathogen. An acid trehalase [EC 3.2.1.28] was purified to homogeneity from a culture of a locust-specific pathogen, Metarhizium anisopliae, and its properties were characterized. The gene (ATM1) of this acid trehalase was also isolated. The pure enzyme can efficiently hydrolyze haemolymph trehalose into glucose in vitro. The new acid trehalase appearing in the haemolymph of Locusta migratoria infected with M. anisopliae had the same pI and substrate specificity as the purified fungal acid trehalase, and the concentration of trehalose in the haemolymph decreased sharply after infection. RT-PCR also revealed the ATM1 gene's expression in the haemolymph of the infected insects. Our results indicated that the acid trehalase may serve as an "energy scavenger" and deplete blood trehalose during fungal pathogenesis.     

Effects of double-stranded RNA in Metarhizium anisopliae var. acridum and Paecilomyces fumosoroseus on protease activities, conidia production, and virulence

Isogenic strains (with and without dsRNA) of the entomogenous fungi Metarhizium anisopliae var. acridum and Paecilomyces fumosoroseus were investigated for correlation between the presence of dsRNA and the production of cuticle-degrading proteases that play an important role in host parasitism, total secreted protein, and conidia production. Similar levels of cuticle-degrading subtilisin-like (Pr1) protease were observed for isogenic strains of M. anisopliae var. acridum after growth in medium supplemented with the cuticle of the grasshopper Rhammatocerus schistocercoides. Similarly, no statistical differences were observed for protease production, detected using the chromogenic substrate azocasein. For P. fumosoroseus isogenic strains, no significant differences in protease activity were observed after growth in the presence of either Euschistus heros or Nezara viridula (Hemiptera: Pentatomidae) cuticle. Similarly, no statistical differences were observed in virulence against E. heros. A comparison of mean conidia production showed a significantly higher production in the dsRNA-free isogenic strains of M. anisopliae var. acridum. Although, for most of the fungal phenotypes analysed, no overt effects were associated with the presence of these dsRNA infections, the reduction in conidia production by the isogenic strains of M. anisopliae var. acridum with dsRNA suggested that it may not be entirely accurate to describe these infections as latent.     

Differential expression of genes involved in entomopathogenicity of the fungi Metarhizium anisopliae var. anisopliae and M. anisopliae var. acridum (Clavicipitaceae)

Expression analysis of the genes involved in germination, conidiogenisis and pathogenesis of Metarhizium anisopliae during its saprophytic and pathogenic life stages can help plan strategies to increase its efficacy as a biological control agent. We quantified relative expression levels of the nitrogen response regulator gene (nrr1) and a G-protein regulator of genes involved in conidiogenesis (cag8), using an RT-qPCR assay. Comparisons were made between M. anisopliae var. anisopliae and M. anisopliae var. acridum during germination and conidiogenesis and at different stages of pathogenesis. The cag8 gene was repressed during germination and induced during conidial development and the pathogenic phase, and the nrr1 gene was induced during germination, conidiogenesis and the pathogenic phase. Both genes were more expressed in M. anisopliae var. anisopliae, demonstrating that different varieties of M. anisopliae differ in activation of genes linked to virulence for certain environments and hosts. This suggests that differences among these varieties in the ability to adapt could be attributed not only to specific genomic regions and genes, but also to differential gene expression in this fungus, modulating its ability to respond to environmental stimuli.     

绿僵菌侵染对椰心叶甲酚氧化酶活性的影响

对椰心叶甲Brontispa longissima(Gestro)成虫血淋巴中酚氧化酶的特性进行分析,并研究绿僵菌(Metarhizium anisopliae)侵染对血浆甲酚氧化酶活性的影响。结果显示,椰心叶甲成虫的血浆及血细胞裂解液中均检测到酚氧化酶活性,且昆布多糖及胰蛋白酶可显著提高其活性。绿僵菌MA-4侵染组在侵染后第1至第5d的血浆酚氧化酶活性高于未侵染组(P<0.05),但是椰心叶甲成虫体内注射10μg昆布多糖后,侵染组的酚氧化酶活性显著低于未侵染组(P<0.05),表明绿僵菌一方面对可激活椰心叶甲的酚氧化酶原激活系统,另一方面又可抑制昆布多糖对椰心叶甲酚氧化酶原激活系统的诱导作用。     

Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria.

The effects of relative humidity (RH) and temperature on the sporulation of Metarhizium anisopliae var. acridum on mycosed cadavers of desert locust, Schistocerca gregaria, were assessed in the laboratory. Quantitative assessments of conidial production over 10 days under constant conditions showed that sporulation was optimized at RH > 96% and at temperatures between 20 and 30 degrees C. Under both these conditions >10(9) conidia/cadaver were produced. At 25 degrees C, conidial yield was maximized under conditions in which cadavers remained in contact with damp substrate. Relatively little sporulation occurred at 15 degrees C (< 3 x 10(7) conidia/cadaver) and 40 degrees C (< 4 x 10(6) conidia/cadaver) and no sporulation occurred at 10 or 45 degrees C. Following incubation, conidial yield was closely related to the water content of locust cadavers. In separate tests, locust cadavers were incubated for 10 days under diurnally fluctuating temperature and RH that comprised favorable (25 degrees C/100% RH) alternating with unfavorable (40 degrees C/80% RH) conditions for sporulation. In this case, fewer conidia were produced compared with cadavers that were incubated under the favorable conditions for an equal period cumulatively but were not periodically exposed to unfavorable conditions. However, this reduced sporulation observed with the fluctuating condition was not observed when cadavers were similarly incubated under favorable/unfavorable conditions of temperature but were not periodically exposed to the low RH condition. This result implies that sporulation is a dynamic process, dependent not only on periodic exposure to favorable RH but also on the interrelation of this with low RH. Associated tests and the monitoring of changes in cadaver weights imply that the mechanism driving the reduced sporulation under fluctuating RH is the net water balance of cadavers, i.e. the cumulative ability of the fungus/cadaver to adsorb water necessary for sporulation at high RH is restricted by water loss associated with intermittent exposure to a low RH. The duration of daily exposure to high humidity appears to be a crucial constraint to the recycling ability of M. anisopliae var. acridum.