Antimicrobial Peptides (AMP) in the Cell-Free Culture Media of Xenorhabdus budapestensis and X. szentirmaii Exert Anti-Protist Activity against Eukaryotic Vertebrate Pathogens including Histomonas meleagridis and Leishmania donovani Species.

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

XENOFOOD-An Autoclaved Feed Supplement Containing Autoclavable Antimicrobial Peptides-Exerts Anticoccidial GI Activity, and Causes Bursa Enlargement, but Has No Detectable Harmful Effects in Broiler Cockerels despite In Vitro Detectable Cytotoxicity on LHM Cells.

Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues.

Interactions between Entomopathogenic Fungi and Entomopathogenic Nematodes.

Entomopathogenic fungi and entomopathogenic nematodes are globally distributed soil organisms capable of infecting and killing a vast variety of insects. Therefore, these organisms are frequently used as biocontrol agents in insect pest management. Both entomopathogenic fungi and nematodes share the soil environment and thus can infest and compete for the same insect host; however, natural co-infections are rarely found due to the cryptic soil environment. Our current knowledge on their interactions within hosts mainly comes from laboratory experiments. Because of the recent trend of combining biocontrol agents to increase their efficacy, many studies have focused on the co-application of different species of EPF and EPNs against various insect pests with variable outcomes ranging from synergistic effects and additive effects to antagonism. In addition, the effect on the development and reproduction of each pathogen varies from normal reproduction to exclusion, and generally the outcomes of the interactions are dependent on pathogen and host species, pathogen doses, and the timing of infection. The present review aims to summarize the current knowledge on the interactions of entomopathogenic fungi and nematodes within an insect host and to estimate the possible effects of the interactions on natural pathogen populations and on their use in biocontrol.

Integrated Biological Control of the Sugar Beet Weevil Asproparthenis punctiventris with the Fungus Metarhizium brunneum: New Application Approaches.

The mass occurrence of the sugar beet weevil (Asproparthenis punctiventris, previously Bothynoderes punctiventris) has been endangering sugar beet cultivation in Austria for centuries. Exacerbated by climatic and political changes (warmer, drier spring and limited access to chemical pesticides), new approaches are needed to counter the problem. The aim of our work was to test whether the bioinsecticide Metarhizium brunneum Ma 43 (formerly M. anisopliae var. anisopliae BIPESCO 5/F52) can be used as a sustainable plant protection product against the sugar beet weevil. Our goal was to control the pest in all its development stages through multiple applications. Therefore, GranMetTM-P, a granular formulation of M. brunneum Ma 43, was applied in spring to establish the fungus in the soil, whereas GranMetTM-WP, a liquid formulation of the production strain, was used in early summer on trap ditches and leaves to target the adult weevils. Soil and plant samples as well as weevils were collected during the planting season from the trial sites to evaluate the development of the fungus and the mycosis of the treated weevils. In addition, data on hibernating weevils and their emigration from untreated field sites was collected. In all field sites, the Metarhizium spp. abundance increased above the background level (<1000 CFU g−1 soil dry weight) after application of the product. With an increasing number of treatments per plot, and thus an increased contact possibility between pest and the fungus, a rise in the mycosis rate was observed. In conclusion, the various Metarhizium application strategies, which are already available or in testing, must be implemented to ensure control in both old and new sugar beet fields. Metarhizium is a further asset in the successful control of this sugar beet pest.

Entomopathogenic nematodes and their symbiotic bacteria: from genes to field uses.

The term "microbial control" has been used to describe the use of microbial pathogens (bacteria, viruses, or fungi) or entomopathogenic nematodes (EPNs) to control various insect pest populations. EPNs are among the best biocontrol agents, and major developments in their use have occurred in recent decades, with many surveys having been conducted all over the world to identify EPNs that may have potential in the management of insect pests. For nematodes, the term "entomopathogenic" means "causing disease to insects" and is mainly used in reference to the bacterial symbionts of Steinernema and Heterorhabditis (Xenorhabdus and Photorhabdus, respectively), which cause EPN infectivity. A compendium of our multiannual experiences on EPN surveys and on their collection, identification, characterization, and use in agro-forestry ecosystems is presented here to testify and demonstrate once again that biological control with EPNs is possible and offers many advantages over chemicals, such as end-user safety, minimal damage to natural enemies, and lack of environmental pollution, which are essential conditions for an advanced IPM strategy.

Steinernema Africanum n. Sp. (Rhabditida, Steinernematidae), a New Entomopathogenic Nematode Species Isolated in the Republic of Rwanda.

Alternatives to hazardous insecticides are urgently needed for an environmentally friendly and effective management of insect pests. One such option is the use of entomopathogenic nematodes (EPN). To increase the availability of EPN with potential for biocontrol, we surveyed agricultural soils in the Republic of Rwanda and collected two Steinernema isolates. Initial molecular characterization showed that they represent a new species, for which we propose the name S. africanum n. sp. To describe this new species, we reconstructed phylogenetic relationships, calculated sequence similarity scores, characterized the nematodes at the morphological level, conducted crossing experiments, and isolated and characterized their symbiotic bacteria. At the molecular level, S. africanum n. sp. is closely related to S. litorale and S. weiseri. At the morphological level, S. africanum n. sp. differs from closely related species by the position of the nerve ring and also because the stoma and pharynx region is longer. The first-generation males have ventrally curved spicules with lanceolate manubrium and fusiform gubernaculum and the second-generation males have rounded manubrium and anteriorly hook-like gubernaculum. Steinernema africanum n. sp. does not mate or produce fertile progeny with any of the closely related species.

Involvement of a novel Pseudomonas protegens strain associated with entomopathogenic nematode infective juveniles in insect pathogenesis.

BACKGROUND: The bioinsecticidal action of entomopathogenic nematodes (EPNs) typically relies on their symbiosis with core bacteria. However, recent studies highlighted the possible involvement of other noncore species. We have recently isolated a novel Pseudomonas protegens strain as a major agent of septicaemia in larvae of the wax moth, Galleria mellonella, infected with a soil-dwelling Steinernema feltiae strain. The actual role of this bacterium in entomopathogenesis was investigated. RESULTS: The association of P. protegens with nematodes appeared to be robust, as supported by its direct and repeated isolation from both nematodes and insect larvae infected for several consecutive generations. The bacterium appeared to be well-adapted to the insect haemocoel, being able to proliferate rapidly after the injection of even a small amount of living cells [100 colony forming units (CFU)] to a larva, causing its fast death. The bacterium also was able to act by ingestion against G. mellonella larvae [median lethal concentration (LC50 ) = 4.0 × 107 CFU mL-1 ], albeit with a slower action, which supports the involvement of specific virulence factors (e.g. chitinases, Fit toxin) to overcome the intestinal barrier to the haemocoel. Varying levels of bacterial virulence were observed on diverse target Diptera and Lepidoptera. CONCLUSION: The soil-dwelling bacterium P. protegens appears to have evolved its own potential as a stand-alone entomopathogen, yet the establishment of an opportunistic association with entomoparasitic nematodes would represent a special competitive advantage. This finding contributes to a deeper understanding of the nematode-bacteria biocontrol agent complex and the deriving paradigm of their use as biological control agents. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Type Strains of Entomopathogenic Nematode-Symbiotic Bacterium Species, Xenorhabdus szentirmaii (EMC) and X. budapestensis (EMA), Are Exceptional Sources of Non-Ribosomal Templated, Large-Target-Spectral, Thermotolerant-Antimicrobial Peptides (by Both), and Iodinin (by EMC).

Antimicrobial multidrug resistance (MDR) is a global challenge, not only for public health, but also for sustainable agriculture. Antibiotics used in humans should be ruled out for use in veterinary or agricultural settings. Applying antimicrobial peptide (AMP) molecules, produced by soil-born organisms for protecting (soil-born) plants, seems a preferable alternative. The natural role of peptide-antimicrobials, produced by the prokaryotic partner of entomopathogenic-nematode/bacterium (EPN/EPB) symbiotic associations, is to sustain monoxenic conditions for the EPB in the gut of the semi-anabiotic infective dauer juvenile (IJ) EPN. They keep pathobiome conditions balanced for the EPN/EPB complex in polyxenic (soil, vanquished insect cadaver) niches. Xenorhabdus szentirmaii DSM16338(T) (EMC), and X. budapestensis DSM16342(T) (EMA), are the respective natural symbionts of EPN species Steinernema rarum and S. bicornutum. We identified and characterized both of these 15 years ago. The functional annotation of the draft genome of EMC revealed 71 genes encoding non-ribosomal peptide synthases, and polyketide synthases. The large spatial Xenorhabdus AMP (fabclavine), was discovered in EMA, and its biosynthetic pathway in EMC. The AMPs produced by EMA and EMC are promising candidates for controlling MDR prokaryotic and eukaryotic pathogens (bacteria, oomycetes, fungi, protozoa). EMC releases large quantity of iodinin (1,6-dihydroxyphenazine 5,10-dioxide) in a water-soluble form into the media, where it condenses to form spectacular water-insoluble, macroscopic crystals. This review evaluates the scientific impact of international research on EMA and EMC.

Biological Control and Insect Pathology.

Agro-forestry intensification is one of the main drivers of the global biodiversity crisis and decline in arthropods and particularly insects [...].

Molecular Characterization and Functional Analysis of the Hb-hsp90-1 Gene in Relation to Temperature Changes in Heterorhabditis bacteriophora.

Understanding how entomopathogenic nematodes respond to temperature changes and have adapted to the local environment is crucial to improve their potential as biocontrol agents. In order to improve understanding of Heterorhabditis bacteriophora's potential adaptability to future climate changes, full-length cDNA and the corresponding gene of heat shock protein 90 (Hsp90) were isolated and fully characterized. The reproductive potential of the Apulian strain of H. bacteriophora increased when the temperature rose from 23 to 30°C, but no reproduction was found at 12°C. Expression analyses revealed that Hb-hsp90-1 was differentially expressed in Infective Juveniles (IJs) and adults (hermaphrodites, females and males). Up-regulation of Hb-hsp90-1 was higher during the recovery process in Galleria mellonella larvae than adults, thus confirming the protective role of Hb-hsp90-1 in coping with the host environment. Silencing of Hb-hsp90-1 resulted in a significant reduction (76%) in the expression level. Silenced IJs took longer than untreated nematodes to infect G. mellonella, showing that Hb-hsp90-1 could be also involved in chemosensation. Furthermore, the number of adults and IJs recovered from G. mellonella infected with silenced nematodes and incubated at 30°C was higher than that obtained from G. mellonella infected with untreated nematodes. These data confirm the crucial role of Hb-hsp90-1 allowing acclimation to increased temperatures and modulation of the recovery process.

Evaluation of Indigenous Entomopathogenic Nematodes as Potential Biocontrol Agents against Popillia japonica (Coleoptera: Scarabaeidae) in Northern Italy.

The natural presence of entomopathogenic nematodes (EPNs) has been investigated in the Piedmont region (Northern Italy) in areas infested by the Japanese beetle Popillia japonica. Thirty-nine out of 155 soil samples (25.2%) were positive for EPNs. Most of the samples contained only steinermatids (92.3%), 5.1% contained heterorhabditids, and one sample (2.6%) contained both genera. All the recovered isolates were identified at species level both morphologically and molecularly. Steinernema carpocapsae was the most abundant and it was mainly distributed in open habitats, such as perennial meadows, uncultivated soils, and cropland, characterized by sandy loam soil texture and acidic pH. Steinernema feltiae has been found associated mainly with closed habitats such as coniferous and deciduous woodland, characterized by sandy loam-texture and extremely acidic soil. The three isolates of Heterorhabditis bacteriophora were collected only in open habitats (perennial meadows and uncultivated fields) characterized by strongly acidic soils with sandy loam texture. The virulence of all EPN natural strains was evaluated by laboratory assays against P. japonica third-instar larvae collected during two different periods of the year (spring, autumn). The results showed that larval mortality was higher for pre-wintering larvae than post-wintering ones. The five more promising EPN isolates were tested in the semi-field assay in which H. bacteriophora natural strains have been shown to be more efficient in controlling P. japonica grubs. All of these results are finally discussed considering the use of these natural EPNs as biological control agents against P. japonica, within an eco-friendly perspective of management.

Molecular profiling of nematode associates with Rhynchophorus ferrugineus in southern Italy.

A survey of nematodes associated with the red palm weevil Rhynchophorus ferrugineus was conducted in southern Italy in 2015 and 2016 in order to create a species inventory and obtain data about nematode biodiversity. A total of 70 insect samples (pupae and adults) were collected from infested Phoenix canariensis, Phoenix dactylifera, and Chamaerops humilis palms in three Italian Regions: sampling took place at 11 locations in Apulia, 1 in Basilicata, and 1 in Sardinia regions. Individual insects were dissected to determine nematode presence, and different nematode species were also recovered from red palm weevil cocoons collected at the sites in Apulia. Individual nematodes were molecularly identified by sequencing the ITS, D2-D3 expansion domains of the 28SrRNA gene and the mitochondrial COI and inferring the phylogenetic relationships. The insect-associated nematofauna identified belonged to the families Rhabditidae, Cephalobidae, and Diplogastridae. Just two nematode species, Teratorhabditis synpapillata and Mononchoides macrospiculum, were always found in association with adult insects and cocoons taken from all sampling sites. This paper reports on the biodiversity of the nematodes associated with R. ferrugineus and on current knowledge of the specific habitat of specialized and divergent entomophilic nematodes.

Comments on the morphology and biology of Pammene castanicola Trematerra & Clausi, 2009 (Lepidoptera: Tortricidae: Grapholitini).

Many insects are reported to damage cultivated chestnut (Castanea sativa Mill.; Fagaceae) in Italy, but the most important pests of chestnut fruits are weevils (Curculio elephas Gyll. and Curculio glandium Marsh.; Curculionidae) and tortricid moths (Pammene and Cydia; Tortricidae) (Pollini, 1998, Speranza, 1999). The larvae of the tortricid pests develop internally, tunnelling in the fruits and eating the endocarp (Rotundo et al. 1991), which significantly reduces nut quality and commercial value. The most damaging tortricid moths in the Italian chestnut industry are Pammene fasciana (Linnaeus) (the early chestnut moth), Cydia fagiglandana (Zeller) (the intermediate chestnut moth), and Cydia splendana (Hübner) (the late chestnut moth) (Pedrazzoli et al. 2012). Pammene castanicola Trematerra & Clausi, 2009 was recently described from chestnut woods near Etna Vulcan, Sicily (Trematerra and Clausi 2009), and it is known only from the vicinity of the type locality. It is similar to Pammene fasciana in morphological and biological features, and it was previously misidentified as P. fasciana. We provide observations on the biology and larval development and present detailed descriptions and illustrations of the mature larva of P. castanicola, with comments on the differences between P. castanicola and P. fasciana.

Activity changes of antioxidant and detoxifying enzymes in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae infected by the entomopathogenic nematode Heterorhabditis beicherriana (Rhabditida: Heterorhabditidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis are lethal parasites of many insect species. To investigate defensive mechanisms towards EPNs in relation to antioxidative and detoxifying enzymes, we chose Tenebrio molitor (Coleoptera: Tenebrionidae) as experimental insect. We studied the activity changes of superoxide dismutases (SODs), peroxidases (PODs), and catalases (CATs), as well as tyrosinase (TYR), acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S-transferase (GSTs) for 40 h in T. molitor larvae infected with Heterorhabditis beicherriana infective juveniles (IJs) at 5 rates (0, 20, 40, 80, and 160 IJs/larva). We found that when T. molitor larvae infected with H. beicherriana at higher rates (80 and 160 IJs/larva), SOD activity quickly increased to more than 70 % higher than that control levels. The activities of POD and CAT increased after 24 h. TYR activity increased slowly at lower rates of infection for 16 h, followed by a slight decrease, and then increasing from 32 to 40 h. The other detoxifying enzymes (GST, CarE, and AChE) were enhanced at lower infection rates, but were inhibited at higher rates. Our results suggested that host antioxidative response and detoxification reactions played a central role in the defensive reaction to EPNs, and that this stress which was reflected by the higher level enzymes activity contributed to the death of hosts. Further study should explore the exact function of these enzymes using different species of EPNs and investigate the links between enzyme activity and host susceptibility to EPNs.

Oscheius onirici sp. n. (Nematoda: Rhabditidae): a new entomopathogenic nematode from an Italian cave.

Oscheius onirici sp. n. (Nematoda: Rhabditidae) was isolated from a karst cave soil of Central Italy. Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region, the D2-D3 expansion domains of the 28S rRNA gene and the 18S rRNA gene were amplified and sequenced. BLAST search at NCBI by using all molecular markers revealed that this taxon is similar to Oscheius species. Phylogenetic trees of ITS, 28S and 18S rDNA revealed that O. onirici sp. n. belongs to Dolichura-group. Oscheius onirici sp. n. is characterized by small body size and stoma rhabditoid type. Female reproductive system is amphidelphic. Males are rare with peloderan bursa, spicules slender and small, nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern. Entomopathogenicity bioassay revealed that this nematode is capable of infecting larvae of Galleria mellonella and Tenebrio molitor.

Mosquitoes LTR retrotransposons: a deeper view into the genomic sequence of Culex quinquefasciatus.

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements.The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking "master copy" elements from which they could have originated. The features of the repetitive sequences found in these non-autonomous LTR retrotransposons are described, and their possible role discussed.These results integrate the existing data on the genomics of an important virus-borne disease vector.

Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy).

A new Steinernema species was isolated from three different sandy soil samples along the Platamona Beach, in the north-west coast of Sardinia Island (Italy). This new species is characterized by the following morphological characters: infective third-stage juvenile with a body length of 866+/-61 (767-969)microm, distance from head to excretory pore of 63+/-2.7 (59-68)microm, tail length of 81+/-3.2 (76-89)microm, ratio E (%) 77+/-3.4 (68-83); male tail with a mucron only in the second generation, spicule length of 66+/-1.4 (64-67)microm and gubernaculum length of 44+/-1.4 (43-46)microm in the first generation male; female of first generation with a slight vulval protrusion and ratio D (%) of 53+/-4.0 (47-63). The new species differs distinctly from the related species (S. feltiae, S. kraussei, S. litorale, S. oregonense and S. cholashanense) in some morphometric values such as percentage of hyaline portion, ratios of gubernaculum/spicule length, spicule head length/width. The DNA analyses of the internal transcribed spacers and D2D3 regions show that the studied nematode isolates are a new species. Cross hybridisation tests with S. feltiae, S. kraussei, S. litorale, S. weiseri and S. oregonense showed that these species were reproductively isolated.