Conidiobolus lunulus, a newly discovered entomophthoralean species, pathogenic and specific to leaf-cutter ants.

Conidiobolus lunulus is a recently described entomophthoralean species isolated from leaf-cutter ants. This fungus discharges not only primary but also secondary conidia and microconidia of different shapes. Because nothing was known about the biology of the fungus, and its interactions with hosts, we first evaluated if its pathogenicity against leaf-cutter ants changes with the fungal age (time grown in vitro), and if it is related to the conidial structures produced. Afterwards, we tested its virulence at three combinations of temperature and relative humidity. In addition, we noted all visible causes of death by recovering different microorganisms from the dead, non-sterilized, ants to evaluate C. lunulus virulence when pathogens carried naturally by the ants were present. Finally, we used the conditions that lead to the highest mortality to evaluate fungal virulence to other host species, including non-leaf-cutter ants. Results indicated that C. lunulus was pathogenic from a culture age of 1 to 5 days, with a peak at 2-days-old, from which we registered median lethal times of 1-2 days and 85% of the cadavers with fungal conidiation. Our results suggest that primary conidia and moon-shaped microconidia were infective. Evaluations of mortality using 2-days-old cultures on several leaf-cutter ant colonies showed 1) significantly faster mortality of C. lunulus inoculated ants in comparison to controls, 2) significantly greater and faster mortality at 23.7 °C than at 21.2 °C, 3) significantly higher and faster mortality at 88% than at 57% RH, and 4) a significant reduction of other pathogens in C. lunulus inoculated ants in comparison to controls. C. lunulus was highly specific to leaf-cutter ants, as hardly any increase in mortality was observed on inoculated ants, and no conidia were recorded on cadavers of the other three non-leaf-cutter ant species tested. Our results highlight that C. lunulus is a very promising biological control agent against leaf-cutter ants.

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus.

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.

Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus.

The entomopathogenic fungus Conidiobolus coronatus produces enzymes that may hydrolyze the cuticle of Galleria mellonella. Of these enzymes, elastase activity was the highest: this figure being 24 times higher than NAGase activity 553 times higher than chitinase activity and 1844 times higher than lipase activity. The present work examines the differences in the hydrolysis of cuticles taken from larvae, pupae and adults (thorax and wings), by C. coronatus enzymes. The cuticles of the larvae and adult thorax were the most susceptible to digestion by proteases and lipases. Moreover, the maximum concentration of free N-glucosamine was in the hydrolysis of G. mellonella thorax. These differences in the digestion of the various types of cuticle may result from differences in their composition. GC-MS analysis of the cuticular fatty acids isolated from pupae of G. mellonella confirmed the presence of C 8:0, C 9:0, C 12:0, C 14:0, C 15:0, C 16:1, C 16:0, C 17:0, C 18:1, C 18:0, with C 16:0 and C 18:0 being present in the highest concentrations. Additional fatty acids were found in extracts from G. mellonella imagines: C 10:0, C 13:0, C 20:0 and C 20:1, with a considerable dominance of C 16:0 and C 18:1. In larvae, C 16:0 and C 18:1 predominated. Statistically significant differences in concentration (p≤0.05) were found between the larvae, pupae and imago for each fatty acid. The qualitative and quantitative differences in the fatty acid composition of G. mellonella cuticle occurring throughout normal development might be responsible for the varied efficiency of fungal enzymes in degrading larval, pupal and adult cuticles.

Transcriptomic insight into pathogenicity-associated factors of Conidiobolus obscurus, an obligate aphid-pathogenic fungus belonging to Entomopthoromycota.

BACKGROUND: Conidiobolus obscurus is a widespread fungal entomopathogen with aphid biocontrol potential. This study focused on a de novo transcriptomic analysis of C. obscurus. RESULTS: A number of pathogenicity-associated factors were annotated for the first time from the assembled 17 231 fungal unigenes, including those encoding subtilisin-like proteolytic enzymes (Pr1s), trypsin-like proteases, metalloproteases, carboxypeptidases and endochitinases. Many of these genes were transcriptionally up-regulated by at least twofold in mycotized cadavers compared with the in vitro fungal cultures. The resultant transcriptomic database was validated by the transcript levels of three selected pathogenicity-related genes quantified from different in vivo and in vitro material in real-time quantitative polymerase chain reaction (PCR). The involvement of multiple Pr1 proteases in the first stage of fungal infection was also suggested. Interestingly, a unique cytolytic (Cyt)-like δ-endotoxin gene was highly expressed in both mycotized cadavers and fungal cultures, and was more or less distinct from its homologues in bacteria and other fungi. CONCLUSION: Our findings provide the first global insight into various pathogenicity-related genes in this obligate aphid pathogen and may help to develop novel biocontrol strategy against aphid pests. © 2018 Society of Chemical Industry.

[A case of rhinofacial entomophthoromycosis in Soudano-Sahelian tropical climate in Burkina Faso]. / Un cas d'entomophthoromycose rhinofaciale en climat tropical soudano-sahélien au Burkina Faso.

We describe a rhinofacial entomophthoramycosis case in a sexagenarian (65 years old) housewife. She was immunocompetent and resident of Burkina Faso. She consulted both the service of dermatology and the service of stomatology of the Teaching Hospital of Bobo-Dioulasso in February 2016 for a diffuse facial tumefaction evolving over six months. This tumefaction was associated with headaches and a left nasal obstruction. Histological examination of the lesion showed an important and polymorphic inflammatory reaction. Also, a filamentous fungus with wide non-septated hyphae and right-angled fungal branching, consistent with mucormycosis was isolated. Mycological diagnosis based on fungal culture with Sabouraud medium without any antibiotic and cyclohexemide after incubation at 27°C and at 30°C was negative. Furthermore, it was not possible to amplify the DNA extracted from biopsy. Antifungal therapy based on the administration of fluconazole per os at 800mg/day was started allowing clinical improvement. This is the first case of a rhinofacial entomophtharomycosis documented in Bobo-Dioulasso. Rhinofacial entomophthoromycosis is largely unknown, even in tropical regions such as Burkina Faso. This lack of knowledge results in a delay in the diagnosis, and subsequently a bad prognosis. It is therefore urgent to improve knowledge on this disease to guide diagnostic steps, prognosis of outcome, and antifungal therapy.

Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus.

Entomopathogenic fungi infect insects via penetration through the cuticle, which varies remarkably in chemical composition across species and life stages. Fungal infection involves the production of enzymes that hydrolyse cuticular proteins, chitin and lipids. Host specificity is associated with fungus-cuticle interactions related to substrate utilization and resistance to host-specific inhibitors. The soil fungus Conidiobolus coronatus (Constantin) (Entomophthorales: Ancylistaceae) shows virulence against susceptible species. The larvae and pupae of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae), Calliphora vomitoria (Linnaeus), Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Musca domestica (Linnaeus) (Diptera: Muscidae) are resistant, but adults exposed to C. coronatus quickly perish. Fungus was cultivated for 3 weeks in a minimal medium. Cell-free filtrate, for which activity of elastase, N-acetylglucosaminidase, chitobiosidase and lipase was determined, was used for in vitro hydrolysis of the cuticle from larvae, puparia and adults. Amounts of amino acids, N-glucosamine and fatty acids released were measured after 8 h of incubation. The effectiveness of fungal enzymes was correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations suggest compounds used by the fungus as nutrients, whereas negative correlations may indicate compounds responsible for insect resistance. Adult deaths result from the ingestion of conidia or fungal excretions.

The composition of the cuticular and internal free fatty acids and alcohols from Lucilia sericata males and females.

GC, GC-MS, and HPLC-LLSD analyses were used to identify and quantify cuticular and internal lipids in males and females of the blow-fly (Lucilia sericata). Sixteen free fatty acids, seven alcohols and cholesterol were identified and quantitatively determined in the cuticular lipids of L. sericata. Cuticular fatty acids ranged from C(6) to C(20) and included unsaturated entities such as 16:1n-9, 18:1n-9, 20:4n-3 and 20:5n-3. Cuticular alcohols (only saturated and even-numbered) ranged from C(12) to C(20) in males and C(10) to C(22) in females. Only one sterol was found in the cuticular lipids of both males and females. 23 free fatty acids, five alcohols and cholesterol were identified in the internal lipids. Internal fatty acids were present in large amounts-7.4 mg/g (female) and 10.1 mg/g (male). Only traces of internal alcohols (from C(14) to C(26) in males, from C(14) to C(22) in females) were found in L. sericata. Large amounts of internal cholesterol were identified in L. sericata males and females (0.49 and 0.97 mg/g of the insect body, respectively).

Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus.

Eighteen fatty acids identified in the cuticle of three insect species representing differing susceptibilities to C. coronatus infection, were tested for effects on the in vitro growth and pathogenicity of the parasitic fungus. At all applied concentrations (0.1-0.0001% w/v) growth was inhibited by C(16:0), C(16:1), C(18:0), C(18:1), C(18:2), C(18:3), C(20:0) and C(20:1). At high concentrations spore germination was inhibited by C(7:0), C(8:0), C(9:0), C(10:0), C(12:0), C(18:2) and C(18:3) and hyphal growth was merely retarded by C(5:0), C(6:0), C(6:2), C(14:0), C(16:0), C(16:1), C(18:0,) C(18:1), C(20:0) and C(20:1). The presence of C(15:0) at the 0.1% concentration stimulated growth of C. coronatus. Sporulation was inhibited by all concentrations of C(16:0) and C(18-20) fatty acids. Low concentrations of C(5:0), C(6:0), C(6:2) and C(7:0) enhanced sporulation. Fatty acids C(5-12) as well as C(18:3), C(20:0) and C(20:1) decreased the ability of fungal colonies to infect G. mellonella while C(16:1) elevated it thus suggesting that C(16:1) may stimulate production of enzymes involved in the host invasion. Toxicity of metabolites released into incubation medium decreased with varying degrees in the presence of C(6:0), C(6:2,) C(7:0), C(9:0), C(12:0), C(16:1), C(18:2), C(18:3), C(20:0) and C(20:1); other fatty acids had no effect. Further work is needed to analyse the effects of exogenous fatty acids on the C. coronatus enzymes implicated in fungal pathogenicity as well as on the production of insecticidal metabolites.

A new thermophilic species of Conidiobolus from India.

A Conidiobolus isolate growing optimally at 40 degrees C was isolated from decomposing leaf litter and has been designated as a new species, Conidiobolus thermophilus. Colony growth, conidial discharge and smooth zygospore formation was rapid at 40 degrees C, while comparative growth at 35 and 45 degrees C was slower. On the basis of its thermophilic character and morphological distinctness from all other species, the isolate is considered as a species new to science. There have been no published reports of any thermophilic or thermotolerant strains of Conidiobolus. The present fungus was isolated as part of an ongoing programme of selective isolation of unusual/rare thermophilic fungi from compost and decomposed terrestrial plant materials.

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection.

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.

Opportunism of Conidiobolus obscurus stems from depression of infection in situ to progeny colonies of host alatae as disseminators of the aphid-pathogenic fungus.

Conidiobolus (Entomophthorales: Ancylistaceae) includes common aphid pathogens but causes sporadic mycosis worldwide. This epizootiological opportunism was explored herein by examining the potential of mycosis transmission in the progeny colonies of 513 Myzus persicae alates as disseminators of C. obscurus often infecting aphids. The alates exposed to spore showers were flown for 2.05 (0.01-8.95) km on flight mills and then reared individually on cabbage at 20-23 degrees C for 14 day colonization. All truly infected alates were mycosed within 6 days and averagely left 3.9 (0-15) nymphs while those uninfected produced 11.6 (0-35) nymphs during the same period. Secondary and tertiary infections occurred only in 16.2% and 4.8% of the progeny colonies of the mycosed alates respectively, due to c. 60% of the cadavers forming resting spores. Most of the contagious infections appeared on days 4-8 after colonization and no more occurred from day 11 onwards. Trends of colony sizes (last-day averaging 51.5 aphids) and mycosis transmission (sixth-day maximum 6.3%) fit well to logistic (r2 = 0.99) and Gompertz growth models (r2 = 0.91) respectively. The results confirm that the opportunism of C. obscurus stems from depression of contagious infection after dissemination by host alates and suggest that it be overwhelmed by the prevalence of other non-resting fungal species.

The influence of Conidiobolus coronatus on phagocytic activity of insect hemocytes.

An essential component of the insect cellular response is phagocytosis. Analyses of the in vitro phagocytosis could be useful for the studies of the relationship between insects and their pathogens. Fungal metabolites are known to inhibit phagocytosis whereas components of the fungal cell wall stimulate phagocytosis. To achieve a better understanding of fungal pathogenesis in insects, haemocyte populations of two insect species susceptible to Conidiobolus coronatus infection (Galleria mellonella, Dendrolimus pini ) were compared with haemocytes of the resistant species (Calliphora erythrocephala ). Fungal infection increased phagocytic activity of G. mellonella plasmatocytes 3.3 times and this of D. pini plasmatocytes 2.1 times. Analysis of infected C. erythrocephala larvae did not reveal any influence of C. coronatus upon phagocytic activity.

First description of the disease by Conidiobolus osmodes on Tipula paludosa larvae with the report of a natural epizootic.

A new fungal pathogen of Tipula paludosa (Tipulidae: Diptera) larvae, Conidiobolus osmodes (Ancylistaceae: Entomophthorales), was found during a survey of tipulid larval pathogens in Northumbria and Cumbria in England in 1997-1999. The fungus caused an epizootic in a population at Close House during autumn 1999 and spring 2000 with prevalence rising fourfold reaching about 40% in April 2000. The disease development was presented and the fungus was described from naturally infected larvae and artificial cultures.