Rileyi Infection Research Articles

An entomopathogenic fungus exploits its host humoral antibacterial immunity to minimize bacterial competition in the hemolymph

BackgroundThe insect hemolymph (blood-equivalent fluid), composed of a large number of hemocytes (blood cells) and a variety of soluble immune effectors, is hostile for pathogens including fungi. In order to survive in the insect hemocoel (body cavity), the entomopathogenic fungus (EPF) has evolved two classical coping strategies, namely evasion and suppression of the host immune reactions. However, it remains unclear whether EPF has other ways of coping with host immunity.ResultsIn this study, we demonstrated that Metarhizium rileyi (an EPF) infection by injection of blastospores into the hemocoel enhanced the plasma antibacterial activity of cotton bollworm (Helicoverpa armigera), which was partially due to the enhanced expression of antimicrobial peptides (AMPs). The early stage of M. rileyi infection induced the translocation of gut bacteria into the hemocoel, where they were subsequently cleared due to the enhanced plasma antibacterial activity. Further, we showed that the enhanced plasma antibacterial activity and AMP expression were attributable to M. rileyi but not the invasive gut bacteria (opportunistic bacteria). Elevated ecdysone (major steroid hormone in insects) levels in the hemolymph at 48 h post-M. rileyi infection might contribute to the enhanced expression of AMPs. The fungus-elicited AMPs, such as cecropin 3 or lebocin, exhibited potent inhibitory activity against the opportunistic bacteria but not against hyphal bodies. In addition, the opportunistic bacteria competed with hyphal bodies for amino acid nutrients.ConclusionsM. rileyi infection induced the translocation of gut bacteria, and then the fungi activated and exploited its host humoral antibacterial immunity to eliminate opportunistic bacteria, preventing them from competing for nutrients in the hemolymph. Unlike the classical strategies, EPF utilizes to evade or suppress host immunity, our findings reveal a novel strategy of interaction between EPF and host immunity.BbkogtQagejkWT1MfyqrGJVideo

Identification and analysis of C-type lectins from Helicoverpa armigera in response to the entomopathogenic fungus Metarhizium rileyi infection

C-type lectins (CTLs) function as pattern recognition receptors (PRRs) and play an important role in the innate immunity of insects. To investigate the role of CTLs in the antifungal responses, we analyzed expression profiles of 36 CTLs of Helicoverpa armigera in the tissues (hemocytes, fat bodies, and midgut) after the infection by entomopathogenic fungus Metarhizium rileyi. The expression levels of many HaCTLs were found to be up-regulated after the infection. Four recombinant HaCTLs (rHaCTL11, rHaCTL12, rHaCTL27, and rHaCTL45) were expressed and purified. Analysis of the purified rHaCTLs revealed that rHaCTLs were able to bind to conidia and hyphal bodies of M. rileyi, and the affinity of rHaCTL11 and rHaCTL27 for hyphal bodies was weaker than for conidia. All these rHaCTLs agglutinate conidia and hyphal bodies in a calcium (Ca2+) dependent manner. Sugar specificity assays showed that d-trehalose, mannan, β-1,3-glucan, d-galactose, glucose, d-raffinose, lipopolysaccharide, and d-xylose can inhibit the binding of HaCTLs to M. rileyi. Additionally, survival assays showed that pretreatment of fungal conidia with rHaCTL11 significantly reduced the rate of host death, and knockdown of HaCTL11 significantly increased H. armigera sensitivity to fungal infection. These results suggest that HaCTLs play significant role as PRRs in the defense of H. armigera against M. rileyi infection.

Comparative genomic analysis of C-type lectin-domain genes in seven holometabolous insect species

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura, and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction.

Sarcocystis rileyi emerging in Hungary: is rice breast disease underreported in the region?

Reports of Sarcocystis rileyi-like protozoa ('rice breast disease') from anseriform birds had been rare in Europe until the last two decades, when S. rileyi was identified in northern Europe and the UK. However, despite the economic losses resulting from S. rileyi infection, no recent accounts are available on its presence (which can be suspected) in most parts of central, western, southern and eastern Europe. Between 2014 and 2019, twelve mallards (Anas platyrhynchos) were observed to have rice breast disease in Hungary, and the last one of these 12 cases allowed molecular identification of S. rileyi, as reported here. In addition, S. rileyi was molecularly identified in the faeces of one red fox (Vulpes vulpes). The hunting season for mallards in Hungary lasts from mid-August to January, which in Europe coincides with the wintering migration of anseriform birds towards the south. Based on this, as well as bird ringing data, it is reasonable to suppose that the first S. rileyi-infected mallards arrived in Hungary from the north. on the other hand, red foxes (Vulpes vulpes), which are final hosts of S. rileyi, are ubiquitous in Hungary, and our molecular finding confirms an already established autochthonous life cycle of S. rileyi in the region. Taken together, this is the first evidence for the occurrence of S. rileyi in Hungary and its region.

The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera.

In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte-mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph.

Gene cloning, expression, and function analysis of SpL14-3-3ζ in Spodoptera litura and its response to the entomopathogenic fungus Nomuraea rileyi.

The 14-3-3 proteins, a highly evolutionarily conserved and ubiquitous protein family in eukaryotic cells, have a range of biological functions including regulation of signal transduction, stress response, apoptosis, and control of the cell cycle. To investigate the function of 14-3-3 in Spodoptera litura, the full length of 14-3-3ζ was cloned from S. litura on the basis of an expressed sequence tag of 14-3-3ζ from the S. litura fat body suppression subtractive hybridization library, and named SpL14-3-3ζ. SpL14-3-3ζ cDNA was 1196 bp with an open reading frame of 744 bp, encoding 247 amino acids. Multiple alignment analysis revealed the putative amino acids shared >80% homology with 14-3-3ζ from other organisms and shared typical conservative structures. Phylogenetic analysis confirmed SpL14-3-3ζ was closely related to other available Lepidoptera 14-3-3ζ. Real-time PCR analysis indicated SpL14-3-3ζ was expressed throughout the developmental stages of S. litura, with a relatively high expression level in pre-pupa, and was expressed constitutively in all examined tissues with relatively high levels in hemocytes and midgut. Moreover, the transcription level of SpL14-3-3ζ could be induced by Nomuraea rileyi infection, up-regulated in hemocytes, followed by head, fat body and midgut. Knocking down SpL14-3-3ζ transcripts by RNAi significantly increased S. litura sensitivity to fungal infection, and resulted in higher mortality of S. litura during the larval development. These results provide novel insights into the 14-3-3ζ signal regulation which may be related to host defense as well as larval development in S. litura.

Structure and expression of a cysteine proteinase gene from Spodoptera litura and its response to biocontrol fungus Nomuraea rileyi

Cysteine proteinases (Cyps) play vital roles in many biological processes, including physiological and pathological reactions. In the present study, we cloned a full cDNA of SlCyp, encoding a 344-amino-acid protein from Spodoptera litura. The putative amino acid sequence shared >75% identity with Cyps from other insects. A phylogenetic analysis revealed that SlCyp is closely related to other known lepidopteran Cyps. Real-time PCR and Western blotting analyses showed that SlCyp is induced by Nomuraea rileyi infection in all the tissues tested. The strongest SlCyp mRNA and protein expression was found in haemocytes, followed by the fat bodies, of unchallenged and N. rileyi-challenged S. litura. A time-course analysis showed that SlCyp mRNA and protein expression levels were upregulated in the haemocytes and fat bodies by N. rileyi infection. Upon N. rileyi infection, the proteolytic activities of SlCyp were also significantly higher in the haemolymph than in normal or phosphate-buffered-saline-challenged controls. These results suggest that SlCyp plays an important role in the innate immunity of S. litura in response to N. rileyi. SlCyp mRNA and protein expression and activities were also elevated during sixth-instar moulting and metamorphosis. Knocking down SlCyp transcripts with double-stranded RNA interference caused prepupal, pupal, and adult phenotypic changes, and SlCyp-silenced mutant larvae displayed a significantly lower survival rate after N. rileyi infection. These facts suggest that SlCyp plays a significant role in resisting N. rileyi infection and an essential role in larval development. Our data should facilitate the development of techniques for S. litura control.

Identification and expression analysis of QM-like gene from Spodoptera litura after challenge by the entomopathogenic fungus Nomuraea rileyi.

A partial sequence of QM homologue was isolated from a Spodoptera litura fatbody suppression subtractive hybridization library. The full-length Spodoptera litura QM ( SpLQM ) cDNA of 838 bp contains a 5′ untranslated region (UTR) of 112 bp, a 3′ UTR of 66 bp, and an open reading frame of 660 nucleotides coding for a 219 amino acid peptide with a molecular weight of 25.5 kDa. Analysis of SpLQM sequence revealed the presence of characteristic motifs, including the ribosomal protein L10 signature and SH3-binding motif. Multiple alignment analysis revealed that SpLQM shares an overall identity of 57.1–99.1% with other members of QM family. Phylogenetic analysis confirmed that SpLQM is closely related to other insect QMs. Analysis of the tissue expression pattern showed that the SpLQM mRNA was expressed in all tissues tested, with highest levels measured in hemocytes, followed by fat bodies. Upon Nomuraea rileyi challenge, SpLQM showed significant upregulation in fat bodies and hemocytes, while slightly upregulation in midguts. The results suggest that SpLQM might play an important role in the innate immunity of S. litura in response to N. rileyi infection. SpLQM was also successfully overexpressed in Escherichia coli, and the recombinant fusion protein SpLQM-His has a molecular weight of 32 kDa.

Thioredoxin peroxidase gene is involved in resistance to biocontrol fungus Nomuraea rileyi in Spodoptera litura: Gene cloning, expression, localization and function

Thioredoxin peroxidases (Tpxs) are a ubiquitous family of antioxidant enzymes that play important roles in protecting organisms against oxidative stress. Here, one Tpx was cloned from Spodoptera litura named as SlTpx. The full-length cDNA consists of 1165bp with 588bp open reading frame, encoding 195 amino acids. The putative amino acid sequence shared >70% identity with Tpxs from other insects. Phylogenetic analysis revealed that SlTpx is closely related to other available lepidopteran Tpxs. Real-time PCR analysis showed that SlTpx can be induced by Nomuraea rileyi infection in some detected tissues at the mRNA level. The strongest expression was found in hemocytes of unchallenged and N. rileyi-challenged S. litura. Western blotting showed SlTpx protein in the hemocytes, head and cuticle from normal S. litura. However, when N. rileyi was inoculated into the body cavity of S. litura larvae, SlTpx protein was detected in head, hemocytes, fatbody, midgut, malpighian tubule, but not in the hemolymph and cuticle. Moreover, time-course analysis showed that SlTpx mRNA/protein expression levels were up-regulated in the hemocytes, when S. litura were infected by N. rileyi or injected with H2O2. The levels of N. rileyi-induced reactive oxygen species (ROS) in hemocytes were evaluated, and revealed that N. rileyi infection caused generation of ROS, and induced changes in expression of SlTpx. In addition, the heterologously expressed protein of this gene in Escherichia coli showed antioxidant activity; it removed H2O2 and protected DNA. Knocking down SlTpx transcripts by dsRNA interference resulted in accelerated insect death with N. rileyi infection. This is believed to be the first report showing that SlTpx has a significant role in resisting oxidative stress caused by N. rileyi infection.

Protozoan parasites from genus Sarcocystis and their investigations in Lithuania

The representatives of the genus Sarcocystis are cyst forming coccidian protozoa parasites broadly prevalent in mammals, birds and reptiles. Sarcocystis parasites are characterized by an obligatory prey-predator two-host life cycle. Currently, over 220 Sarcocystis species are known. Some of Sarcocystis species are pathogenic organisms dangerous to humans, domestic and wild animals. A harmful effect of Sarcocystis mainly occurs in intermediate hosts and depends on the species of parasite, infection intensity and localization in the body. In Lithuania, Sarcocystis species forming sarcocysts in rodents, even-toed ungulates and birds were mainly studied. The ecology and diversity of Sarcocystis parasites were investigated using traditional morphological, DNA analysis methods and transmission experiments. Lithuanian scientists have described and named five Sarcocystis species, i. e. S. rodentifelis, S. wobeseri, S. cornixi, S. anasi, S. albifronsi; for the first time proved Sarcocystis rileyi infection in Europe; established definitive hosts of some Sarcocystis species.

Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi

Nomuraea rileyi is an important pathogenic fungus that can successfully control Spodoptera litura. However, little is known on how S. litura responds to N. rileyi infection. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from the S. litura fat body and the up-regulated genes were identified to isolate differentially expressed genes in response to N. rileyi. A total of 345/1175 random clones screened by cDNA array dot blotting were sequenced, resulting in 117 uniquely expressed sequence tags (ESTs). Potential functional genes were identified by BLAST searches and were categorized into seven groups associated with different biological processes based on the literature and gene ontologies. Among 117 genes, 74 had matches in the non-redundant (NR) protein database and were found to be involved in different biological processes, while 43 of the screened genes were classified to the "unknown function" gene group. Notably, only two genes had previously been reported in S. litura and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 115 genes were found for the first time in S. litura. Semi-quantitative RT-PCR analysis of seven randomly selected genes revealed that most were differentially expressed after N. rileyi infection. qRT-PCR analysis confirmed that four genes (Hsp70, Hsp90, gallerimycin, and cysteine proteinase) were significantly up-regulated after N. rileyi infection. Taken together, the present study identified up-regulated S. litura genes in response to N. rileyi infection. Further investigations are needed to unravel the molecular mechanisms of the genes or proteins potentially involved in the S. litura innate immune defense against N. rileyi infection.

Soybean leaf consumption by Nomuraea rileyi (Fungi: Deuteromycotina)-infected Plathypena scabra (Lepidoptera: Noctuidae) larvae

To determine the impact of Nomuraea rileyi on consumption by the green cloverworm, Plathypena scabra, larvae were reared from eggs obtained from field-collected moths, inoculated with conidia, and placed individually in separate plastic vials with a piece of surface-sterilized soybean leaflet. No significant differences in consumption rates were found between N. rileyi-inoculated and control larvae until after 144 hr post-treatment. After this period, consumption by larvae inoculated as first, second, third, or fourth instars was significantly less than that of control larvae because of mortality. First or second instars inoculated with N. rileyi conidia had significantly longer subsequent stadia than did control larvae. The average LT 50 of all inoculated instar groups was 6.5 days, and there was little evidence of significantly different N. rileyi susceptibility among instar groups. In general during the N. rileyi infection period leading to their deaths, inoculated P. scabra larvae had consumption patterns that were very similar to those of healthy larvae.

Epidemiology of Nomuraea rileyi (Fungi: Deuteromycotina) in Plathypena scabra (Lepidoptera: Noctuidae) Populations from Iowa Soybeans

Data from a 10-year study were analyzed to understand the epidemiology of Nomuraea rileyi (Farlow) Samson in green cloverworm (GCW), Plathypena scabra (F.), populations in Iowa. Larvae were collected by sweeping in soybean fields each year from 1971 through 1980 and were maintained in the laboratory until completion of development or death. Six endemic and 4 outbreak GCW population types were identified from the 10 collection years. N. rileyi was a major agent of GCW mortality, especially during the second GCW generation of outbreak years. The seasonal increase in percentage N. rileyi -caused mortality in outbreak populations is described as a function of the number of days after the date of initial detection of infected larvae. In all years, the mortality rate of the second GCW generation from N. rileyi infection is significantly dependent on the size of the first GCW generation. The date of first prominent (2%) N. rileyi -caused mortality is significantly explained by the accumulated larva-days and accumulated precipitation from 1 June. These results support and supplement existing explanations of GCW population dynamics in Iowa by providing quantitative expressions of N. rileyi epidemiology.

Susceptibility of the Velvetbean Caterpillar and Soybean Looper (Lepidoptera: Noctuidae) to Nomuraea rileyi: Effects of Pathotype, Dosage, Temperature, and Host Age

Comparisons among four Nomuraea rileyi isolates tested against Anticarsia gemmaatalis and Pseudoplusia includens demonstrated distinct differences in both the susceptibility of the two noctuid hosts and the virulence of strains. Host susceptibility to N. rileyi infection was directly influenced by larval age. Maturation of host larvae resulted in increased Lc50 values and in the mean days to death of the N. rileyi strains. The in vivo developmental rate of N. rileyi was affected by the ambient temperature. Temperatures below the calculated optima (26°C) for N. rileyi decreased the developmental rate of the infection process but did not alter the percent N. rileyi-induced mortality of treated A. gemmatalis larvae. A. gemmatalis, with a developmental optimum of 31°C, became increasingly resistant to N. rileyi infection when reared at temperatures above 26°C. The assay and food substrate influenced both the development of the host and the relative virulence of N. rileyi to A. gemmatalis larvae. Comparisons of concentration and time responses demonstrated that larvae assayed on the soybean leaflets were more resistant to N. rileyi infection than larvae assayed on agar substrate and fed artificial diet. TheA. gemmatalis feeding on mature soybean foliage (pod set stage) appeared to be more resistant to N. rileyi infections than did the larvae feeding on prebloom and bloom soybean stages.

Effect of Insecticides, Fungicides, and Insecticide-Fungicide Combinations on Development of Lepidopterous Larval Populations in Soybean 123

The fungicides benomyl, chlorothalonil, thiabendazole, and fentin hydroxide were applied alone and in combination with methyl parathion or methomyl to soybeans. Larval population levels of Plathypena scabra (F.), Anticarsia gemmatalis (Hübner), and Pseudoplusia includens (Walker) were monitored as well as selected arthropod predators and the incidence of the fungal entomopathogens Nomuraea rileyi (Farlow) Samson and Entomophthora gammae (Weiser). Application of fungicides at the recommended rates failed to suppress an epizootic of E. gammae in P. includens populations. The incidence of N. rileyi was suppressed in a low density population of A. gemmatalis . When larval density increased, no difference was detected in the incidence of N. rileyi infection in treated and untreated plots. Differences in predator population levels were not observed in a 1976 P. includens test but generally lower predator populations were noted in the 1977 P. includens-A. gemmatalis test in fungicide and/or insecticide treated plots. Fentin hydroxide alone and treatments which included methyl parathion or methomyl reduced larval populations of P. scabra and A. gemmatalis . Only treatments containing methomyl reduced larval populations of P. includens. Pseudoplusia includens population resurgence was detected following treatments with benomyl and methyl parathion, chlorothalonil + methyl parathion and fentin hydroxide + methyl parathion. Anticarsia gemmatalis population resurgence followed treatment with fentin hydroxide + methomyl.

Pathogenicity of Spicaria rileyi to Pseudoplusia includens

To determine the impact of Nomuraea rileyi on consumption by the green cloverworm, Plathypena scabra, larvae were reared from eggs obtained from field-collected moths, inoculated with conidia, and placed individually in separate plastic vials with a piece of surface-sterilized soybean leaflet. No significant differences in consumption rates were found between N. rileyi-inoculated and control larvae until after 144 hr post-treatment. After this period, consumption by larvae inoculated as first, second, third, or fourth instars was significantly less than that of control larvae because of mortality. First or second instars inoculated with N. rileyi conidia had significantly longer subsequent stadia than did control larvae. The average LT50 of all inoculated instar groups was 6.5 days, and there was little evidence of significantly different N. rileyi susceptibility among instar groups. In general during the N. rileyi infection period leading to their deaths, inoculated P. scabra larvae had consumption patterns that were very similar to those of healthy larvae.