Entomopathogenic nematodes (EPNs) are soil-dwelling parasitic roundworms commonly used as biocontrol agents of insect pests in agriculture. EPN dauer juveniles locate and infect a host in which they will grow and multiply until resource depletion. During their free-living stage, EPNs face a series of internal and environmental stresses. Their ability to overcome these challenges is crucial to determine their infection success and survival. In this review, we provide a comprehensive overview of EPN response to stresses associated with starvation, low/elevated temperatures, desiccation, osmotic stress, hypoxia, and ultra-violet light. We further report EPN defense strategies to cope with biotic stressors such as viruses, bacteria, fungi, and predatory insects. By comparing the genetic and biochemical basis of these strategies to the nematode model Caenorhabditis elegans, we provide new avenues and targets to select and engineer precision nematodes adapted to specific field conditions.

Epithelial hyperplasia and sloughing of the digestive gland in bivalve mollusks are a global phenomenon and occur in species of commercial interest and cultural significance to indigenous peoples. Where hemocytosis, hyperplasia, and necrosis of digestive tubule cells have been observed associated with electron-dense uncoated virus-like particles (VLPs) 25-45 nm in diameter, the condition has been named digestive epithelial virosis (DEV). This condition has been associated with mortalities of some bivalve species in New Zealand. Similar digestive gland alterations, but without detection of associated VLPs, have been reported in other bivalve species worldwide and are termed "DEV-like" since no virus link has been demonstrated. It remains unclear if DEV is an infectious condition and whether associated VLPs are the cause, a contributor, or simply associated with the observed condition. It is also unclear whether DEV or DEV-like conditions pose a biosecurity or economic threat, or alternatively, whether they reflect a natural cyclic event that does not require disease management. In this mini-review, we summarize the history of digestive epithelial alteration with VLPs (i.e., DEV) or without observation of VLPs (i.e., DEV-like), and we examine the evidence for and against viral-like particles as the cause of DEV in bivalves. We also explore other viral afflictions of bivalves and non-infectious agents, such as harmful algae and xenotoxins, that could elicit similar tissue alterations. Future recommendations for approaches to identify key risk factors that lead to the development of digestive epithelial alterations such as DEV include histological characterization of the digestive gland of marine mollusks; the use of metagenome analysis to design primers that could be used for detection of VLPs and to study host microbiota; disease challenges demonstrating that DEV causes pathology and the relationship between DEV intensity and morbidity/mortality.

Avoidance of pathogens and parasites is the first line of defense to survive. Several slug species avoid the parasitic nematode Phasmarhabditis hermaphrodita to reduce infection however, there is nothing known about whether slugs avoid other members of the Phasmarhabditis genus. I exposed two slug species (Deroceras invadens and Limax maculatus) to Phasmarhabditis californica and P. neopapillosa. D. invadens avoided P. californica but was strangely attracted to P. neopapillosa. L. maculatus did not avoid P. californica, but on day 1 and 3 significantly more slugs were found with P. neopapillosa. Reasons for host attraction to P. neopapillosa are discussed.

Wolbachia are known to cause reproductive manipulations and in some arthropod species, Wolbachia were reported to cause changes in gut microbiome. However, the effects of Wolbachia bacteria on the microbiomes of their hosts, including Drosophila flies, have not been fully accessed. Here, we checked the bacterial microbiome in guts of Wolbachia-uninfected and of Wolbachia-infected Drosophila nigrosparsa, both separated into a bleach-only (embryos bleached) and a gnotobiotic (embryos bleached and inoculated with bacteria) treatment. We observed a clear separation between the Wolbachia-infected and the Wolbachia-uninfected samples, and the infected samples had higher variation in alpha diversity than the uninfected ones. There were reductions in the abundances of Proteobacteria (Pseudomonadota), especially Acetobacter, in the infected samples of both treatments. These findings highlight that Wolbachia change the gut microbiome in D. nigrosparsa as well as that the interactions between Wolbachia and bacteria like Acetobacter need to be investigated.

Quercetin is a plant flavonoid with a molecular formula C15H10O7. It has antioxidant, anti-inflammatory, antibacterial, and anti-apoptotic effects in animals. We used red claw crayfish (Cherax quadricarinatus) infected with white spot syndrome virus (WSSV) to investigate quercetin's effects on innate immunity of crustaceans. Quercetin supplementation significantly reduced the mortality of crayfish caused by WSSV infection and the number of VP28 copies in WSSV-infected crayfish. Quantitative real-time PCR analysis showed that dietary quercetin supplementation increased the expression of immune-related genes, like JAK, STAT and ALF. Quercetin supplementation affected the activity of six immune-related enzymes and increased the total number of hemocytes in crayfish. It also significantly reduced the rate of hemocyte apoptosis in both WSSV-infected and uninfected crayfish. These results demonstrate the potential for commercial use of quercetin for the prevention of WSSV disease in crustaceans.

The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features characteristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine- and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

Pigments of conidia play a crucial role in fungal defense against environmental stressors such as UV radiation. The molecular basis of conidial pigmentation has been studied in the entomopathogenic fungus Metarhizium robertsii, while limited information been reported on function mechanisms transcription factors governing conidial pigmentation. Here, we identified transcription factor MrAbaA binding to the promoter regions of both MrPks1 and MrMlac1 in M. robertsii using yeast one-hybrid technology. Chromatin immunoprecipitation quantitative PCR assays further confirmed the interaction. Furthermore, overexpression of MrAbaA in M. robertsii resulted in increased conidial pigment accumulation and enhanced tolerances to UV stress by upregulated the MrPks1 and MrMlac1 expression. Taken together, MrAbaA affects conidial pigmentation by interacting with the promoter regions of both MrPks1 and MrMlac1 in M. robertsii. This work advances the understanding of the regulation mechanism for conidial pigmentation in entomopathogenic fungi.

Cancer-like neoplasms are extremely rarely present in arthropods, particularly in crustaceans. Thus, it is assumed that these animals have some efficient cancer-preventing mechanisms. However, several cases of cancer-like neoplasms are described in crustaceans, though only for the Decapoda. We identified a tumor in the parasitic barnacle Peltogaster paguri (Cirripedia: Rhizocephala), and described its histological structure. A spherical cell mass consisting mostly of roundish cells with big translucent nuclei, prominent nucleoli, and sparse chromatin, and of cells with condensed chromosomes, was found in the main trunk of the P. paguri rootlet system. Numerous mitoses were observed in this area. Such tissue organization is utterly uncharacteristic of the Rhizocephala. Based on acquired histological data, we assume that this tumor is a cancer-like neoplasm. This is the first report of a tumor identified in the rhizocephalans, as well as in non-decapod crustaceans as a whole.

The insect Galleria mellonella is an alternative animal model widely used for studying bacterial infections. It presents a wide range of advantages, including its low cost, easy maintenance and lack of ethical constraints. Among other features, their innate immune system is very similar to that of mammals. In this study, we dissected several larvae infected with important human pathogens: Mycobacterium abscessus, Staphylococcus aureus and Pseudomonas aeruginosa. By observing the fat body, gut, trachea, and hemolymph under the microscope, we were able to describe where bacteria tend to disseminate. We also quantified the number of bacteria in the hemolymph throughout the infection course and found significant differences between the different pathogens. With this work, we aimed to better understand the behavior and dissemination of bacteria in the infected larvae.

For humans, acute and chronic overexposure to ultraviolet (UV) radiation can cause tissue damage in the form of sunburn and promote cancer(s). The immune-modulating properties of UV radiation and health-related consequences are not well known. Herein, we used the larvae of the wax moth Galleria mellonella, to determine UV-driven changes in cellular components of innate immunity. From immune cell (haemocyte) reactivity and the production of antimicrobial factors, these insects share many functional similarities with mammalian cellular innate immunity. After exposing insects to UVA or UVB for up to two hours, we monitored larval viability, susceptibility to infection, haemolymph (blood) physiology and faecal discharge. Prolonged exposure of larvae to UVB coincided with decreased survival, enhanced susceptibility to bacterial challenge, melanin synthesis in the haemolymph, compromised haemocyte functionality and changes in faecal (bacterial) content. We contend G. mellonella is a reliable in vivo model for assessing the impact of UV exposure at the whole organism and cellular levels.

Currently, species within the genus Akanthomyces are poorly studied and explored compared to other hypocrealean entomopathogenic fungi employed as commercial biocontrol agents. This study aimed to molecularly identify 23 Brazilian Akanthomyces strains, most originally isolated from aphids and scales (n = 22), and one from the coffee leaf rust, and further investigate their pathogenicity to six plant-sucking insects as a means to better understand their host spectra. We also explored the capacity of A. muscarius CG935 for blastospore production via liquid fermentation. Akanthomyces dipterigenus, A. muscarius, A. lecanii, and two unidentified species were recognized as naturally occurring in Brazil. Akanthomyces dipterigenus CG829 and A. muscarius CG935 were highly virulent to nymphs of Bemisia tabaci (67.5-85.4% confirmed mortality) and the aphid Aphis fabae (74.6-75.3%), but only the first strain was virulent to the mealybug Planococcus sp. (80.9%). Akanthomyces lecanii CG824 was weakly virulent to all tested insects. None of the strains were pathogenic to the thrips Caliothrips phaseoli, and all strains showed low virulence to the wooly whitefly Aleurothrixus floccosus and the scale Duplachionaspis divergens. Submerged liquid fermentation yields varied from 1.72 × 109 (day 2) to 3.90 × 109 (day 5) blastospores mL-1. Blastospores or aerial conidia from A. muscarius CG935, at a single concentration of 1 × 107 viable propagules mL-1, resulted in 67.5-83.1% mortality of B. tabaci nymphs within 8 days post-treatment. Overall, these results encourage additional studies that could lead to the development of new mycopesticides based on Akanthomyces strains.

An epizootic caused by fungal pathogens occurred among Halyomorpha halys, brown marmorated stink bugs, while they were overwintering, with infections also occurring after overwintering. We report that one of the two pathogens responsible was Colletotrichum fioriniae (Marcelino & Gouli) Pennycook; a species well known as a plant pathogen and endophyte and which has only previously been reported naturally infecting elongate hemlock scales, Fiorinia externa. To prove pathogenicity, H. halys adults challenged with conidia died from infections and the fungus subsequently produced conidia externally on cadavers.

Exotic dung beetles have been introduced to Australia for over 50 years to mitigate issues caused by dung produced by livestock. This study aims at identifying fungi affecting a beetle colony and investigating their source. Fungal hyphae emerging from the cuticle of dead beetles were cultured and a multigene phylogeny showed that Beauveria bassiana and B. australis both infected these introduced beetles, likely acquired through local soil or dung. This is the first record of B. australis infecting family Scarabaeidae. This study highlights the importance of fungal outbreak management in insect rearing before release, and challenges associated with new pathogens.

Hematodinium perezi is a dinoflagellate endoparasitic in marine crustaceans, primarily decapods. It occurs in juvenile blue crabs, Callinectes sapidus, at high prevalence levels and has severe pathogenic effects in this host. The life history outside the host has not been experimentally investigated and, until now, transmission using dinospores has not been successful. We investigated the natural transmission dynamics of H. perezi in the laboratory using small juvenile crabs, which are highly susceptible to infection in the field, and elevated temperatures, which are known to stimulate dinospore production. Natural water-borne transmission to naïve crabs varied between 7 and 100% and was not correlated with dinospore densities measured from their aquaria water. Infections appeared to develop quickly in naïve hosts at 25 °C, suggesting that elevated temperatures as seen in the late summer and early autumn have a strong influence on the transmission of H. perezi in natural systems.

Enterocytozoon hepatopenaei (EHP), an obligate intracellular parasite classified as microsporidia, is an emerging pathogen with a significant impact on the global shrimp aquaculture industry. The understanding of how microsporidia germinate has been a key factor in exploring its infection process. However, the germination process of EHP was rarely reported. To gain insight into the germination process, we conducted a high-throughput sequencing analysis of purified EHP spores that had undergone in vitro germination treatment. This analysis revealed 137 differentially expressed genes, with 84 up-regulated and 53 down-regulated genes. While the functions of some of the genes remain unknown, this study provides important data on the transcriptomic changes before and after EHP germination, which can aid in further studies on the EHP infection mechanism.

Temperature is an important abiotic factor influencing the survival and fitness of pathogens as well as their hosts. We investigated the effect of three temperatures (18 °C, 27 °C and 37 °C) on survival and performance of black soldier fly larvae (BSFL), Hermetia illucens L., upon infection by an entomopathogenic Gram-negative bacterium, Pseudomonas protegens Pf-5. The effect of different temperatures on pathogen fitness was investigated both in vivo and in vitro. Pathogen performance under exposure to the insect antimicrobial peptide cecropin was investigated at the three temperatures using radial-diffusion plate assays. Higher rearing temperatures resulted in higher larval survival, increased larval weight, and higher inhibitory activity of cecropin against P. protegens Pf-5. At higher temperature, bacterial growth, both in vivo and in vitro, was reduced, resulting in increased BSFL survival. These observations collectively indicate the important effect of rearing temperature on host-pathogen interactions and the possibility to apply temperature treatment in reducing entomopathogen effects in BSFL.

Culex pipiens (Diptera: Culicidae) is a vector of many human and animal diseases. Its control is regarded as a preventative approach that is focused on effectively managing such diseases. In this context, dose response assays of two insecticides, bendiocarb and diflubenzuron were performed with two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae against 3rd instar C. pipiens larvae. The most effective agents, combination experiments as well as enzymatic activities of phenoloxidase (PO) and chitinase (CHI) were also assessed. The results showed that diflubenzuron was more effective at low concentrations (LC50: 0.001 ppm) than bendiocarb (LC50: 0.174 ppm), whereas M. anisopliae was more effective (LC50: 5.2 × 105 conidia/mL) than B. bassiana (LC50: 7.5 × 107 conidia/mL). Synergistic interactions were observed when diflubenzuron was applied at 2- and 4-days post- exposure to M. anisopliae, with the highest degree of synergism observed when diflubenzuron was applied 2 days post-fungal exposure (χ2 = 5.77). In contrast, additive interactions were recorded with all other insecticide-fungal combinations. PO activities significantly (p ≤ 0.05) increased during 24 h after a single diflubenzuron treatment as well as when diflubenzuron was applied prior to M. anisopliae, whereas suppressed after 24 h when M. anisopliae applied prior to diflubenzuron as well as after 48 h from single and combined treatments. CHI activity increased 24 h after both single and combined treatments, the activity remained elevated 48 h after a single diflubenzuron treatment and when diflubenzuron was applied after M. anisopliae. Histological study of the cuticle by transmission electron microscopy revealed abnormalities following single and combined treatments. Germination of the conidia and production of the mycelium that colonizes the lysing cuticle was obvious when diflubenzuron was applied 48 h after M. anisopliae exposure. Overall, these results demonstrate that M. anisopliae is compatible with diflubenzuron at lower concentrations and that combined applications can improve C. pipiens management.

A total of 53 anamorphic strains of Brazilian Cordyceps species currently maintained in a government-owned culture collection, were reassessed for diversity and species identity using multi-loci-based phylogenetic methods. The strains used in this study were originally obtained from soil samples or were isolated from insects of the orders Hemiptera, Lepidoptera, Coleoptera and Diptera, mostly from agricultural sites. A Bayesian phylogenetic tree was constructed based on a concatenation of five loci (ITS, LSU, RPB1, RPB2 and TEF). In a few cases of ambiguity, morphological traits were also considered for species delimitations. Considerable variability within the set of strains was detected and six Cordyceps species were identified: C. amoenerosea, C. fumosorosea, C. javanica, C. tenuipes and, for the first time, C. brevistroma and C. spegazzinii are reported in Brazil. Four other taxonomically equivocal groups, closely related to other known taxa (C. amoenerosea, C. cateniannulata, C. polyarthra and C. spegazzinii), were also recognized, although further studies will be required to confirm their identifications or their descriptions as new species. Cordyceps javanica was the most common species in our dataset, originally isolated from soil and several different insect orders, and includes 17 strains from the whitefly, Bemisia tabaci. Interestingly, strains previously identified as C. fumosorosea based on morphology and growth characteristics, were shown to be C. javanica, including the active ingredients of some commercial mycoinsecticides. Cordyceps farinosa, usually mentioned in the literature as occurring in Brazil, was not found in our study. Since most strains were from insect crop pests, further studies with hosts from non-agricultural settings or from environmental samples would be advisable for a deeper understanding of the occurrence of anamorphic Cordyceps in Brazil.

The brown garden snail (Cornu aspersum) is a major agricultural pest, causing damage to a wide range of economically important crops. Withdrawal or restricted use of pollutant molluscicides like metaldehyde has prompted a search for more benign control products. This study investigated the response of snails to 3-octanone; a volatile organic compound (VOCs) produced by the insect pathogenic fungus Metarhizium brunneum. Concentrations of 1 - 1000 ppm of 3-octanone were first assessed in laboratory choice assays to determine behavioural response. Repellent activity was found at 1000 ppm whereas attractance was found for the lower concentrations of 1, 10 and 100 ppm. These three concentrations of 3-octanone were carried forward in field evaluations to assess potential for use in "lure and kill" strategies. The highest concentration (100 ppm) was the most attractive to the snails but also the most lethal. Even at the lowest concentration this compound proved toxic effects making 3-octanone an excellent candidate for the development as a snail attractant and molluscicide.

White Spot Syndrome Virus (WSSV) continues to cause considerable loss to shrimp farmers globally with frequent outbreaks even in specific pathogen free Peneaus vannamei. Our studies showed that the bodyweight (BW) of PL has a bearing on their susceptibility to the virus. To test this hypothesis, PL of the same age group and family were grouped according to BW (10-20, 30-40, and 50-60 mg) and challenged through immersion route with two viral doses (106 and 107 virus copies/L of water). It was observed that the PL became susceptible to WSSV at ≥50 mg BW. In the 50-60 mg PL group, the higher challenge dose shows a sharp mortality curve with 100% mortality at 10 days post immersion, while the lower dose shows a steady increase in cumulative mortality that reaches 100% on the 13th day post immersion. The study also brings out that an in vivo viral load of approximately 3.5 to 4.5 × 107WSSV copies/100 ng shrimp DNA results in mortality. This is the first report on the relationship between BW and WSSV susceptibility in shrimp PL. Also reported here is a quantitative assessment of WSSV infection in P. vannamei PL and an optimized challenge protocol.

Carcinus spp. are global aquatic invaders and carriers of several parasites, including a taxonomically unrecognised microsporidian recently observed from Argentina. We provide genome drafts for two parasite isolates, one from Carcinus maenas and one from Carcinus aestuarii, and use multi-gene phylogenetics and genome comparison methods to outline their similarities. Their SSU genes are 100 % similar and other genes have an average similarity of 99.31 %. We informally name the parasite Agmasoma carcini, terming the isolates Ac. var. aestuarii and Ac. var. maenas, following the wealth of genomic data available for each. This study follows on from Frizzera et al. (2021), where this parasite was first histologically identified.

Wolbachia bacterial endosymbionts provide protection against pathogens in various arthropod species but the underlying mechanisms remain misunderstood. By using a natural Wolbachia nuclear insert (f-element) in the isopod Armadillidium vulgare, we explored whether Wolbachia presence is mandatory to observe protection in this species or the presence of its genes is sufficient. We assessed survival of closely related females carrying or lacking the f-element (and lacking Wolbachia) challenged with the bacterial pathogen Salmonella enterica. Despite marginal significant effects, the f-element alone did not appear to confer survival benefits to its host, suggesting that Wolbachia presence in cells is crucial for protection.

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic virus with a ∼212 kb linear dsDNA genome that encodes 215 putative open reading frames (ORFs). Proteomic analysis has revealed that the IIV6 virion consists of 54 virally encoded proteins. Interactions among the structural proteins were investigated using the yeast two-hybrid system, revealing that the protein of 415R ORF interacts reciprocally with the potential envelope protein 118L and the major capsid protein 274L. This result suggests that 415R might be a matrix protein that plays a role as a bridge between the capsid and the envelope proteins. To elucidate the function of 415R protein, we determined the localization of 415R in IIV6 structure and analyzed the properties of 415R-silenced IIV6. Specific antibodies produced against 415R protein were used to determine the location of the 415R protein in the virion structure. Both western blot hybridization and immunogold electron microscopy analyses showed that the 415R protein was found in virions treated with Triton X-100, which degrades the viral envelope. The 415R gene was silenced by the RNA interference (RNAi) technique. We used gene-specific dsRNA's to target 415R and showed that this treatment resulted in a significant drop in virus titer. Silencing 415R with dsRNA also reduced the transcription levels of other viral genes. These results provide important data on the role and location of IIV6 415R protein in the virion structure. Additionally, these results may also shed light on the identification of the homologs of 415R among the vertebrate iridoviruses.

A new species of entomopathogenic fungus, Metarhizium indicum, which derives its species epithet after its Indian origin is reported here. The fungus was found to cause natural epizootics in leafhopper (Busoniomimus manjunathi) infesting Garcinia gummi-gutta (Malabar tamarind), an evergreen spice tree native to South and Southeast Asia, known for its use as a culinary flavourant, dietary supplement and traditional remedy for various human ailments. The fungus was found to cause more than 60% mortality in field collected insects. The identity of the new species was established based on its distinct morphological characteristics and multi-gene sequence data analyses. Phylogenetic analyses using internal transcribed spacer region (ITS), DNA lyase (APN2) and a concatenated set of four marker genes [translation elongation factor 1-alpha (TEF), β-tubulin (BTUB), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2)] along with marked differences in nucleotide composition and genetic distance unambiguously support our claim that the present fungus infecting Garcinia leafhopper is a new addition to the genus Metarhizium.

Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat to sericulture production, and traditional sanitation practices remain the main strategy for controlling BmNPV infection. Although RNAi targeting BmNPV genes engineered into transgenic silkworms has shown to be a promising approach in reducing viral infection, it cannot block viral entry into host cells. Therefore, there is an urgent need to develop new effective prevention and control measures. In this study, we screened a monoclonal antibody 6C5 that potently neutralizes BmNPV infection by clamping the internal fusion loop of the BmNPVglycoprotein64 (GP64). Furthermore, we cloned the VH and VL fragments of mAb-6C5 from the hybridoma cell, and the eukaryotic expression vector of scFv6C5 was constructed to anchor the antibody on the cell membrane. The GP64 fusion loop antibody-expressing cells exhibited a reduced capacity for BmNPV infection. The results from our study provide a novel BmNPV control strategy and lay the foundation for the future development of transgenic silkworms with improved antiviral efficacy.

Microsporidians (Microsporidia) are a diverse group of obligate and intracellular parasites of eukaryotes. There is evidence that the real species diversity in the phylum could be greatly underestimated, especially for microsporidians parasitic on invertebrates. Mosquitoes (Culicidae) are among very important microsporidian host groups. However, to date, no extensive survey on the prevalence of microsporidians in European mosquitoes has been performed. Here, we used mosquitoes collected in west-central Poland and a metabarcoding approach to examine the prevalence and diversity of microsporidian species among European mosquitoes. We found that up to one-third of mosquitoes in Europe may be infected with at least 13 microsporidian species belonging to the genera Amblyospora, Hazardia, Encephalitozoon, Enterocytospora, and Nosema and the holding genus Microsporidium. The lack of a difference in microsporidian prevalence between mosquito sexes implies that other factors, e.g., temperature or humidity, affect microsporidian occurrence in adult mosquitoes. Each microsporidian species was found in at least three mosquito species, which suggests that these microsporidians are polyxenic rather than monoxenic parasites. The co-occurrence of at least two different microsporidian species was found in 3.6% of host individuals. The abundance of microsporidian DNA sequences suggests interactions between co-occurring parasites; however, these results should be confirmed by microscopic and quantitative methods. In addition, further histological research is required to describe Microsporidium sp. PL01 or match its DNA to that of an already described species.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured shrimp. It is generally believed that the target organs of IHHNV in shrimp include tissues of ectodermal and mesodermal origin, but do not normally include organ systems of endodermal origin, such as hepatopancreas. In this study, the feeding challenge of IHHNV in different organs (pleopods, muscles, gills, and hepatopancreas) of Penaeus vannamei was studied. The PCR results showed that hepatopancreas of P. vannamei had the strongest IHHNV positivity (100% positive, 19.4 copies/mg) in the feeding challenge experiment. Gills and pleopods had similar infectivity to IHHNV (86.7% positive, 10.6 and 10.5 copies/mg). Among the four organs tested in this study, the IHHNV positivity of muscles was the weakest (33.3% positive, 4.7 copies/mg). The IHHNV infection to hepatopancreas of P. vannamei was also histological confirmed. Our current data indicated that the shrimp tissues derived from the endoderm such as hepatopancreas could also be infected by IHHNV.

Winter oilseed rape (Brassica napus) is one of the largest crops in Europe and the cabbage stem flea beetle Psylliodes chrysocephala is one of its major pests. Since the ban of neonicotinoids for seed treatment, farmers apply pyrethroids in autumn to control the cabbage stem flea beetle. Current studies show that the insect develops resistance to this group of chemicals. Biological control with entomopathogenic nematodes (EPNs) represents a possible, environmentally friendly alternative control measure. In the present work, we considered three strategies to control the cabbage stem flea beetle: applying the nematodes against the first larval stage in the soil, against the second and third larval stages inside the plant or against the adult beetles. In laboratory experiments, we found the third larval instar to be the most susceptible stage and the adult beetle the less susceptible one. Steinernema feltiae and the cold active SDT1-IL1 Heterorhabditis bacteriophora strain, with a reduction potential of 89 and 76 %, respectively, proved to be the most virulent EPNs against P. chrysocephala in pot experiments at 15 °C. Moreover, we performed four field trials to test the efficacy of H. bacteriophora and S. feltiae against the larvae. The highest reduction in the field trials was 45% and 39%, obtained with SDT1-IL1 and a mixture of H. bacteriophora and S.feltiae, respectively. The present study provides preliminary information about the potential of EPNs to control P. chrysocephala and represents a start point for the development of a competitive and sustainable alternative to pyrethroids.

The gut microflora plays an important role in insect development and physiology. The gut bacterial microbiome of the fall armyworm (FAW), Spodoptera frugiperda, in both cornfield and laboratory-reared populations was investigated using a 16S metagenomic approach. The alpha- and beta-diversity of the cornfield FAW populations varied among sampling sites and were higher than those of the laboratory-reared FAW population, indicating that different diets and environments influence the gut bacterial composition. To better understand the interaction between the microbiome and entomopathogenic fungi (EPF), FAWs from organic and conventionally managed corn fields and from the laboratory-reared colony were inoculated with Beauveria bassiana NCHU-153 (Bb-NCHU-153). A longer median lethal time (LT50) was observed in the Bb-NCHU-153-infected cornfield FAW population than in the laboratory-reared FAWs. In terms of the microbiome, three Bb-NCHU-153-infected FAW groups showed different gut bacterial compositions compared to noninfected FAW. Further investigation of the cooccurrence network and linear discriminant analysis (LDA) of effect size (LEfSe) revealed that the enriched bacterial genera, such as Enterococcus, Serratia, Achromobacter, and Tsukamurella, in the gut might play the role of opportunistic pathogens after fungal infection; in contrast, some gut bacteria of Methylobacterium, Marinomonas, Paenochrobactrum, Pseudomonas, Acinetobacter, Delftia, Dietzia, Gordonia, Leucobacter, Paracoccus, and Stenotrophomonas might be probiotics against EPF infection. These results indicated that EPF infection can change the gut bacterial composition and lead to a pathobiome in the FAW and that some bacterial species might protect the FAW from EPF infection. These findings could be applied to the design of pathobiome-inducing biocontrol strategies.

The invasive alien ladybird Harmonia axyridis (Coleoptera: Coccinellidae) hosts a wide range of natural enemies. Many observations have been done in nature but experimental studies of interactions of multiple enemies on Ha. axyridis are rare. In light of this knowledge gap, we tested whether the host phenotype and presence of bacterial endosymbionts Spiroplasma and Wolbachia affected parasitism of Ha. axyridis by the ectoparasitic fungus Hesperomyces harmoniae (Ascomycota: Laboulbeniales). We collected 379 Ha. axyridis in the Czech Republic, processed specimens, including screening for He. harmoniae and a molecular assessment for bacteria, and calculated fecundity and hatchability of females. We found that high hatchability rate (71 %) was conditioned by high fecundity (20 eggs daily or more). The average parasite prevalence of He. harmoniae was 53 %, while the infection rate of Spiroplasma was 73 % in ladybirds that survived in winter conditions. Wolbachia was only present in 2 % of the analyzed ladybirds. Infection by either He. harmoniae or Spiroplasma did not differ among host color morphs. In the novemdecimsignata morph, younger individuals (with orange elytra) were more heavily parasitized compared to old ones (with red elytra). Fecundity and hatchability rate of females were unaffected by infection with either He. harmoniae or Spiroplasma. However, female ladybirds co-infected with He. harmoniae and Spiroplasma had a significantly lower fecundity and hatchability compared to females with only one or no symbiont.

A pathogen with high virulence potential in some host species, Perkinsus marinus remains a challenge for the ecological integrity of marine ecosystems and the health of bivalve molluscs. This study investigates the occurrence of P. marinus in Crassostrea sp. in estuaries of the Potengi River and the Guaraíras lagoon in Rio Grande do Norte, Brazil. A total of 203 oyster samples that tested positive for Perkinsus sp. in Ray's fluid thioglycollate medium (RFTM) were subjected to species-specific quantitiative PCR, where 61 animals (30.05 %) presented amplification graphs with a melting temperature of 80.1 ± 0.6 °C matching the positive control. This was the first record of P. marinus in oysters in these estuaries using qPCR as a diagnostic tool.

The gut bacterial microbiota of insects has been shown to play essential roles in processes related to physiology, metabolism, and innate immunity. In this study, we firstly performed a broad analysis of the gut bacteria in Lymantria dispar asiatica, one of the most devastating forestry defoliators. We analyzed the bacterial composition among different individuals from lab-reared or wild-collected using 16 s rRNA-sequencing, revealing that the gut bacteria of wild-collected larvae were highly diverse, while lab-reared larvae were only associated with a few genera. We found Lactobacillus sp. present in all the gut samples, which indicates that it is part of the core microbiome in the caterpillar. Further Beauveria bassiana infection-based assays showed that the mortality of non-axenic L. dispar asiatica larvae was significantly higher than that of axenic larvae at 72 h. Moreover, we isolated several bacteria from the hemolymph of the non-axenic larvae infected by B. bassiana, which may be caused by the translocation of gut bacteria from the gut to the hemocoel. Reintroduction of Enterococcus sp., Pseudomonas sp., Enterobacter sp., and Microbacterium sp. into axenic larvae recurred the larval mortality in their non-axenic counterpart. Taken together, our study demonstrates that the gut bacteria of L. dispar asiatica are highly volatile, and different bacteria taxa can promote host infection by entomopathogenic fungus, providing a new strategy for the pest management.

Mass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by 16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates (FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge. This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 × 107 CFU while a lower dose (3 × 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels.

Spodoptera frugiperda is a pest that poses a serious threat to the production of food and crops. Entomopathogenic fungi, such as Beauveria bassiana, have shown potential for S. frugiperda control. However, the mechanism of this biological control of pathogens is not fully understood, such as how antioxidant enzyme activities and metabolic profiles in S. frugiperda larvae are affected when infected by entomopathogenic fungi. This study assessed the antioxidant enzyme activities and shift in metabolomic profile in the S. frugiperda larvae infected with B.bassiana. The results indicate a pattern of initial increase and subsequent decrease in the activities of superoxide dismutase, catalase, and peroxidase in the B.bassiana-infected larvae. And the enzyme activities at 60 h of infection ended significantly lower than those of the uninfected larvae. A total of 93 differential metabolites were identified in the B.bassiana-infected larvae, of which 41 metabolites were up-regulated and 52 were down-regulated. These metabolites mainly included amino acids, nucleotides, lipids, carbohydrates, and their derivatives. Among the changed metabolites, cystathionine, l-tyrosine, l-dopa, arginine, alpha-ketoglutaric acid, d-sedoheptulose-7-phosphate and citric acid were significantly decreased in B. bassiana-infected larvae. This indicated that the fungal infection might impair the ability of S. frugiperda larvae to cope with oxidative stress, leading to a negative impact of organism fitness. Further analyses of key metabolic pathways reveal that B. bassiana infection might affect purine metabolism, arginine biosynthesis, butanoate metabolism, and phenylalanine metabolism of S. frugiperda larvae. The findings from this study will contribute to our understanding of oxidative stress on immune defense in insects, and offer fundamental support for the biological control of S. frugiperda.

Infections of insects with insect-specific RNA viruses are common and can affect host fitness and health. Previously, persistent RNA virus infections were detected in tephritid fruit flies, including the Queensland fruit fly (Bactrocera tryoni), Australia's most significant horticultural pest. Their transmission modes and efficiency are unclear yet may influence virus epidemiology in field and laboratory populations. Using standard RT-PCR and RT-qPCR we detected iflavirus, cripavirus and sigmavirus in five laboratory populations recently established with field-collected B.tryoni. Virus absence in some individuals suggested that virus transmission is incomplete. Random virus segregation in an isofemale experiment resulted in the establishment of isofemale lines with and without iflavirus and cripavirus. In infected lines, viral loads normalised against host gene transcripts were variable, but did not differ between pupae and adults. Iflavirus and cripavirus were transmitted horizontally, with viruses detected (including at low viral loads) in many previously uninfected individuals after four days, and in most after 12 days cohabitation with infected flies. Iflavirus, but not cripavirus, was transmitted vertically, and surface-sterilised embryos contained high loads. Furthermore, high iflavirus loads in individual females resulted in high loads in their offspring. We demonstrated that viruses are highly prevalent in laboratory populations and that it is possible to establish and maintain uninfected fly lines for the assessment of virus transmission and host effects. This is important for pest management strategies such as the sterile insect technique which requires the mass-rearing of flies, as their fitness and performance may be affected by covert virus infections.

Crayfish have strong ecological impacts in freshwater systems, yet our knowledge of their parasites is limited. This study describes the first systemic microsporidium (infects multiple tissue types) Alternosema astaquatica n. sp. (Enterocytozoonida) isolated from a crayfish host, Faxonius virilis, using histopathology, transmission electron microscopy, gene sequencing, and phylogenetics. The parasite develops in direct contact with the host cell cytoplasm producing mature spores that are monokaryotic and ellipsoid in shape. Spores have 9-10 coils of the polar filament and measure 3.07 ± 0.26 µm (SD) in length and 0.93 ± 0.08 µm (SD) in width. Our novel isolate has high genetic similarity to Alternosema bostrichidis isolated from terrestrial beetles; however, genetic data from this parasite is restricted to a small fragment (396 bp) of the SSU gene. Additional data related to spore morphology and development, host, environment, and ecology indicate that our novel isolate is distinct from A. bostrichidis, which supports a new species description. Alternosema astaquatica n. sp. represents a novel member of the Orthosomella-like group which appears to be a set of opportunists within the Enterocytozoonida. The presence of this microsporidium in F. virilis could be relevant for freshwater ecosystems across this crayfish's broad geographic range in North America and may affect interactions between F. virilis and invasive rusty crayfish Faxonius rusticus in the Midwest USA.

The springtail, Folsomia candida, is a soil arthropod commonly used to evaluate environmental toxins. Conflicting data on the toxicity of the herbicide paraquat prompted re-evaluation of its effects on F. candida survival and reproduction. Paraquat has an LC50 of about 80 μM when tested in the absence of charcoal; charcoal, often used in test arenas to facilitate visualization of the white Collembola, has a protective effect. Survivors of paraquat treatment fail to resume molting and oviposition, suggesting an irreversible effect on the Wolbachia symbiont that restores diploidy during parthenogenetic reproduction of this species.

We document gut parasites in co-occurring Apis, Bombus, and Osmia spp. in the Northern Virginia region, USA. Trypanosomatidea occurred in sixty percent of specimens and 13% carried Nosematidae. We found strong host partitioning: Lotmaria passim and Vairimorpha (Nosema) ceranae predominated in Apis, and Crithidia bombi and V. bombi in Bombus. We did not detect pathogen spread from Apis to Bombus but did detect sharing of C. bombi between Bombus and Osmia, higher parasite levels in Apis at sites with apiaries, and clustering of Vairimopha infection. Given the presence of C. bombi in Osmia, we suggest disease sharing across taxa be monitored.

Several PCR methodologies are available for the detection of Enterocytozoon hepatopenaei (EHP) that target the SSU rRNA gene. However, these methodologies are reported as unsuitable for the detection of EHP due to specificity issues. Here, we report the applicability of two commonly used SSU rRNA methodologies for the detection of additional microsporidia from the genus Vittaforma that is present in cultured Penaeus vannamei from Costa Rica. The molecular detection of DNA of the novel microsporidia can only be achieved using SSU rRNA targeting methodologies and does not cross-react with the highly specific spore wall protein gene PCR detection method.

'Candidatus Liberibacter' is a genus of plant-associated bacteria that can be transmitted by insects of the superfamily Psylloidea. Since many members of this genus are putative causal agents of plant diseases, it is crucial in studying their interactions with the psyllid vectors. However, previous studies have mainly focused on few species associated with diseases of economic significance, and this may potentially hinder the development of a more comprehensive understanding of the ecology of 'Ca. Liberibacter'. The present study showed that an endemic psyllid species in Taiwan, Cacopsylla oluanpiensis, is infected with a species of 'Ca. Liberibacter'. The bacterium was present in geographically distant populations of the psyllid and was identified as 'Ca. Liberibacter europaeus' (CLeu), a species which generally does not induce plant symptoms. Analysis of CLeu infection densities in male and female C. oluanpiensis with different abdominal colors using quantitative polymerase chain reaction revealed that CLeu infection was not significantly associated with psyllid gender and body color. Instead, CLeu infection had a negative effect on the body sizes of both male and female psyllids, which is influenced by bacterial titer. Investigation on CLeu's distribution patterns in C. oluanpiensis's host plant Pittosporum pentandrum indicated that CLeu does not behave as a plant pathogen. Also, results showed that nymph-infested twigs had a greater chance of carrying high loads of CLeu, suggesting that ovipositing females and the nymphs are the main source of the bacterium in the plants. This study is not only the first to formally report the presence of CLeu in C. oluanpiensis and plants in the family Pittosporaceae, but also represents the first record of the bacterium in Taiwan. Overall, the findings in this work broaden the understanding of associations between psyllids and 'Ca. Liberibacter' in the field.

Microsporidia are emerging intracellular parasites of most known animal phyla in all ecological niches. In shrimp aquaculture, the microsporidium Enterocytozoon hepatopenaei (EHP) is a major cause of concern inflicting tremendous losses to shrimp producers in southeast Asia. During a histopathological examination of Penaeus vannamei samples originating in a country from Latin America presenting slow growth, we observed abnormal nuclei in the epithelial cells of the hepatopancreas. A PCR screening of the samples using DNA isolated from paraffin embedded tissues for the SSU rRNA gene of EHP provided a 149 bp amplicon. In situ hybridization using the SSU rRNA gene probe provided a positive signal in the nuclei instead of the cytoplasm. Sequence analysis of the SSU rRNA gene product revealed a 91.3 %, 89.2 % and 85.4 % sequence identity to Enterocytozoon bieneusi, E. hepatopenaei and Enterospora canceri respectively. Furthermore, phylogenetic analysis revealed the newly discovered microsporidium clustered with E. bieneusi. Considering the intranuclear location of the novel microsporidium and the differences in the sequence of the SSU rRNA, we tentatively consider this parasite a new member of the genus Enterospora sp. The pathogenicity and distribution of the shrimp Enterospora sp. are currently unknown. Our future efforts are focused on the characterization and development of diagnostic tools for this parasite to understand if it acts as an emergent pathogen that might require surveillance to prevent its spread.

Locusts occasionally represent a danger in Africa despite intensive management measures, leading to severe yield loss and a commensurate loss of food and money. The laboratory assessment of the toxicity of the Photorhabdus luminescens bacteria and its cell-free filtrate used Schistocerca gregaria nymphs in the second and fifth nymphs as test insects. Greater mortality was seen in locust nymphs of the second and fifth instars due to the high levels of toxicity produced by the bacterial suspension and its cell-free filtrate. The amounts of protein, fat, and carbohydrates in the treated locusts were drastically reduced. For the treated second and fifth instar nymphs of the desert locust, adverse effects on the muscular layers of the midgut and the muscles in the jumping legs were investigated.

Cordyceps fumosorosea is an entomopathogenic fungus with a global distribution and is used for the biological control of agricultural pests. High conidial productivity and tolerance to abiotic stresses such as elevated temperature and ultraviolet radiation (UV-B) are desired characteristics in candidate isolates for commercial products. Our goal in this study was to characterize promising isolates of C. fumosorosea from five Brazilian biomes regarding conidial production, tolerance to UV-B, and elevated temperature (45°). Seventy-two isolates out of 172 were chosen visually, based on growth and sporulation in culture medium, and grown on parboiled rice. Next, fourteen isolates were selected, based on productivity on rice and origin of isolation, for production in polypropylene bags and submitted to UV-B for 2, 4, 6, and 8 h or to 45 °C for 30, 60, and 90 min. High variations in conidial production were observed among isolates, and a positive correlation was observed between UV-B and heat tolerance. The isolates ESALQ4556 and ESALQ4778 showed the highest yields of conidial production in polypropylene bags (3.51 × 109 conidia/g dry rice), while ESALQ1296, an isolate recovered from insects, was the most tolerant to UV-B and 45 °C. Exposure to radiation for more than 4 h and placed directly at 45 °C for more than 30 min significantly reduced conidial germination for all C. fumosorosea isolates. These results contribute to a better understanding of the tolerance to abiotic factors of Brazilian isolates of C. fumosorosea.

Ticks are carriers of viruses that can cause disease in humans and animals. The longhorned ticks (Haemaphysalis longicornis; LHT), for example, mediates the severe fever with thrombocytopenia syndrome virus (SFTSV) in humans, and the population of ticks is growing due to increases in temperature caused by climate change. As ticks carry primarily RNA viruses, there is a need to study the possibility of detecting new viruses through tick virome analysis. In this study, viruses in LHTs collected in Korea were investigated and virus titers in ticks exposed to the entomopathogenic fungus Metarhizium anisopliae JEF-290 were analyzed. Total RNA was extracted from the collected ticks, and short reads were obtained from Illumina sequencing. A total of 50,024 contigs with coding capacity were obtained after de novo assembly of the reads in the metaSPAdes genome assembler. A series of BLAST-based analyses using the GenBank database was performed to screen viral contigs, and three putative virus species were identified from the tick meta-transcriptome, such as Alongshan virus (ALSV), Denso virus and Taggert virus. Measurements of virus-expression levels of infected and non-infected LHTs failed to detect substantial differences in expression levels. However, we suggest that LHT can spread not only SFTSV, but also various other disease-causing viruses over large areas of the world. From the phylogenetic analysis of ALSV glycoproteins, genetic differences in the ALSV could be due to host differences as well as regional differences. Viral metagenome analysis can be used as a tool to manage future outbreaks of disease caused by ticks by detecting unknown viruses.

Aedes aegypti, an important vector of viral diseases affecting humans in the tropics, generally oviposits just above the water line of small artificial bodies of water. Within the first hours after being deposited eggs are highly susceptible to desiccation, and the chorion undergoes profound processes of sclerotization. Most uneclosed eggs remain viable for months, and their susceptibility to entomopathogenic fungi turns them into reasonable targets for focal control strategies. This study explored the sensitivity of newly deposited eggs to Metarhizium humberi IP 46 conidia. Immediate exposure of eggs oviposited onto a wet, conidium-treated substrate or application of conidia onto eggs within the first 72h after deposition revealed no clearly higher ovicidal effect caused by pre-germinating or germinating conidia or by further fungal development during this initial phase of chorionic sclerotization and embryogenesis than occurs on fully sclerotized eggs. Fungal application techniques, whether direct or indirect, seemed to matter little at the low concentrations applied here; using higher conidial concentrations of the entomopathogen might yield greater mortality of eggs regardless of their physiological age. Quite apart from the data on the biocontrol potential of M. humberi against A. aegypti eggs, these studies demonstrate that the bleaching of highly melanized egg chorions allows detailed visualization of early events of pathogenic fungal attachment, germination, penetration, and initial development inside a target insect.

Besides killing insects, Metarhizium spp. have shown another ecological role as plant associates. Partial genra and groups of these entomopathogenic fungi act as plant growth promoters during root colonization. Here, we report that Metarhizium robertsii produces a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD encoded by MracdS, MrACCD), which is involved in promoting early vegetative growth in wheat, while Metarhizium acridum lacks a functional ACCD, although a gene encoding for a putative ACCD has been identified in its genome. MracdS expression was up-regulated by a max 10.7-fold with 3 mM ACC and high ACCD enzymatic activities were induced by either ACC (7.5-fold) or wheat root (3.2-fold). In contrast, no ACCD activity was detected in M. acridum in the presence of both inducers. In pot assay, wheat seeds were treated with wild-type M. robertsii (Mr23), wild-type M. acridum (Mac324), MracdS disruption mutant (ΔMracdS) and M. acridum transformant harboring heterologous MracdS (Mac324-MracdS). Relative to the control seeds treated with heat-killed conidia, Mr23, ΔMracdS and Mac324-MracdS increased root length (by 66.2, 31.8 and 40.2%), and plant biomass (by 56.6, 42.1 and 40.9%). Nevertheless, ΔMracdS deficient in ACCD activity heavily impaired its capability of wheat growth promotion by decrease of 20.7% in root length relative to Mr23. In addition, Mr23 and Mac324-MracdS also increased shoot growth (by 42.3, and 42.7%) while ΔMracdS failed. Mac324 showed no effect on plant growth during the test. These data suggest a role for ACCD in the plant growth promotion effect by M. robertsii, which is irrelevant to Metarhizium colonization of roots since rhizosphere competency of both Mr23 and Mac324 are unaffected by the change of ACCD activity.

Topical applications of insecticides β-cyfluthrin, imidacloprid, and spinosad in combination with Beauveria bassiana (topical and contact filter paper application) induced synergistic interactions in lesser mealworm larvae, increasing mortality and in some cases numbers of larval cadavers exhibiting conidiogenesis. Reduced concentrations (LC10, LC20, LC30) of the insecticides induced sublethal effects in lesser mealworm larvae, inhibiting development (mass, head-capsule width, moulting) after eight days' exposure and movement behaviour (area explored and distance travelled) after 3 h' exposure. The most potent synergist was ß-cyfluthrin, it strongly inhibited larval development and movement while significantly increasing mortality and conidiogenesis in B. bassiana-infected larvae. Imidacloprid also strongly inhibited larval development and movement, but only produced weak short-lived synergistic increases in mortality, with no increase in conidiogenesis. Spinosad induced no effect on development and limited effect on movement, but still induced moderate short-lived synergistic increases in mortality and conidiogenesis. Intoxicated larvae exposed to B. bassiana on filter paper for 3 h showed no synergistic interactions, except when intoxicated by spinosad.

Formulation technology has been the primordial focus to improve the low viability and erratic infectivity of entomopathogenic nematodes (EPNs) for foliar application. Adaptability to the fluctuating environment is a key trait in ensuring the survival and efficacy of EPNs. Hence, tailoring formulations towards EPNs foliar applications would effectively deliver consistent and reliable results for above-ground applications. EPNs survival and activity were characterized in novel Pickering emulsion post-application in planta cotton foliage. Two different types of novel formulations, Titanium Pickering emulsion (TPE) and Silica Pickering emulsion Gel (SPEG), were tailored for EPNs foliar applications. We report an extension of survival and infectivity to 96 hrs under controlled conditions by SPEG formulations for survival of IJ's on cotton foliage. In addition, survival of IJs (LT50) was extended from 14hrs in water to > 80 hrs and > 40 hrs by SPEG and TPE respectively. SPEG accounted for the slowest decrease of live IJs per surface area in comparison to TPE and control samples over time, exhibiting a 6-fold increase at 48 hrs. Under extreme conditions, survival and efficacy were extended for 8hrs in SPEG compared to merely 2hrs in control. Potential implications and possible mechanisms of protection are discussed.

Heritable microbes that exhibit reproductive parasitism are common in insects. One class of these are the male-killing bacteria, which are found in a broad range of insect hosts. Commonly, our knowledge of the incidence of these microbes is based on one or a few sampling sites, and the degree and causes of spatial variation are unclear. In this paper, we examine the incidence of the son-killer microbe Arsenophonus nasoniae across European populations of its wasp host, Nasonia vitripennis. In preliminary work, we noticed two female N. vitripennis producing highly female biased sex ratios in a field study from the Netherlands and Germany. When tested, the brood from Germany was revealed to be infected with A. nasoniae. We then completed a broad survey in 2012, in which fly pupal hosts of N. vitripennis were collected from vacated birds' nests from four European populations, N. vitripennis wasps allowed to emerge and then tested for A. nasoniae presence through PCR assay. We then developed a new screening methodology based on direct PCR assays of fly pupae and applied this to ethanol-preserved material collected from great tit (Parus major) nests in Portugal. These data show A. nasoniae is found widely in European N. vitripennis, being present in Germany, the UK, Finland, Switzerland and Portugal. Samples varied in the frequency with which they carry A. nasoniae, from being rare to being present in 50% of the pupae parasitised by N. vitripennis. Direct screening of ethanol-preserved fly pupae was an effective method for revealing both wasp and A. nasoniae infection, and will facilitate sample transport across national boundaries. Future research should examine the causes of variation in frequency, in particular testing the hypothesis that N. vitripennis superparasitism rates drive the variation in A. nasoniae frequency through providing opportunities for infectious transmission.

Stingless bees are important social corbiculate bees, fulfilling critical pollination roles in many ecosystems. However, their gut microbiota, particularly the fungal communities associated with them, remains inadequately characterised. This knowledge gap hinders our understanding of bee gut microbiomes and their impacts on the host fitness. We collected 121 samples from two species, Tetragonula carbonaria and Austroplebeia australis across 1200 km of eastern Australia. We characterised their gut microbiomes and investigated potential correlations between bee gut microbiomes and various geographical and morphological factors. We found their core microbiomes consisted of the abundant bacterial taxa Snodgrassella, Lactobacillus and Acetobacteraceae, and the fungal taxa Didymellaceae, Monocilium mucidum and Aureobasidium pullulans, but variances of their abundances among samples were large. Furthermore, gut bacterial richness of T. carbonaria was positively correlated to host forewing length, an established correlate to body size and fitness indicator in insects relating to flight capacity. This result indicates that larger body size/longer foraging distance of bees could associate with greater microbial diversity in gut. Additionally, both host species identity and management approach significantly influenced gut microbial diversity and composition, and similarity between colonies for both species decreased as the geographic distance between them increased. We also quantified the total bacterial and fungal abundance of the samples using qPCR analyses and found that bacterial abundance was higher in T. carbonaria compared to A. australis, and fungi were either lowly abundant or below the threshold of detection for both species. Overall, our study provides novel understanding of stingless bee gut microbiomes over a large geographic span and reveals that gut fungal communities likely not play an important role in host functions due to their low abundances.

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages. In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

Subtropical Florida blue crabs, Callinectes sapidus, exhibit differing life history traits compared to their temperate counterparts, likely influencing symbiont infection dynamics. Little information exists for Florida C. sapidus symbiont profiles, their distribution among various habitats, and influence on crab condition. Using histopathology, genomics, and transmission electron microscopy, we describe the first symbiont profiles for Florida C. sapidus occupying freshwater to marine habitats. Twelve symbiont groups were identified from 409 crabs including ciliophorans, digenean, microsporidian, Haplosporidia, Hematodinium sp., Nematoda, filamentous bacteria, gregarine, Callinectes sapidus nudivirus, Octolasmis sp., Cambarincola sp., and putative microcell. Overall, 78% of C. sapidus were documented with one or more symbiont groups demonstrating high infection rates in wild populations. Environmental variables water temperature and salinity explained 48% of the variation in symbiont groups among Florida habitats, and salinity was positively correlated with C. sapidus symbiont diversity. This suggests freshwater C. sapidus possess fewer symbionts and represent healthier individuals compared to saltwater populations. Crab condition was examined using the reflex action mortality predictor (RAMP) to determine if reflex impairment could be linked to symbiont prevalence. Symbionts were found positively correlated with crab condition, and impaired crabs were more likely to host symbionts, demonstrating symbiont inclusion may boost predictive ability of the RAMP application. The microsporidian symbiont group had a particularly strong effect on C. sapidus reflex response, and impairment was on average 1.57 times higher compared to all other symbiont groups. Our findings demonstrate the importance of considering full symbiont profiles and their associations with a spatially and temporally variable environment to fully assess C. sapidus population health.

Pollinators have experienced significant declines in the past decade, in part due to emerging infectious diseases. Historically, studies have primarily focused on pathogens in the Western honey bee, Apis mellifera. However, recent work has demonstrated that these pathogens are shared by other pollinators and can negatively affect their health. Here, we surveyed honey bees and 15 native bee and wasp species for 13 pathogens traditionally associated with honey bees. The native bee and wasp species included 11 species not previously screened for pathogens. We found at least one honey bee-associated pathogen in 53% of native bee and wasp samples. The most widely distributed and commonly detected pathogens were the microsporidian Nosema ceranae, the bacterium Melissococcus plutonius, and the viruses deformed wing virus and black queen cell virus. The prevalence of viruses was generally higher in honey bees than in native bees and wasps. However, the prevalence of M. plutonius and the brood fungus Ascosphaera apis was significantly higher in some native bee species than in honey bees. The data also reveal novel trends in the association between co-occurring pathogens in honey bees and native bees and wasps at the pathogen community level. These results can inform the assessment of risks that native pollinator species face from pathogen stress, and indicate that many non-viral pathogens, notably M. plutonius and N. ceranae, are far more widely distributed and commonly found in native bees and wasps than previously thought.

Enterocytozoon hepatopenaei (EHP) is a kind of microsporidian parasite belonging to fungi, and poses a serious threat to prawn farmers. Due to the lack of effective treatments for EHP, the establishment of a rapid and sensitive detection method would be beneficial to the control and prevention of this prawn parasitic disease. In this study, an isothermal enzymatic recombinase amplification (EHP-ERA) assay that could diagnose EHP within 20 min at 42 °C was developed and evaluated. The determined final concentrations of primers and probe in the reaction system were 400 nM and 120 nM, respectively. EHP-ERA was carried out within 13 min (24.31 ± 0.37 Ct) with a detection limit of 10 copies/μL. The results of specificity test showed that EHP-ERA had no cross-reactivity with white spot syndrome virus (WSSV), Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (VpAHPND), and infectious hypodermal and hematopoietic necrosis virus (IHHNV) and specific pathogen free (SPF) shrimp. Using 32 clinical samples, the practical diagnostic results of EHP-ERA was consistent with nested PCR and real-time PCR (qPCR) under the premise of less time-consuming and simpler operation. In summary, we established a simple, rapid, and effective ERA assay for the detection of EHP, which had great potential to be widely used in both lab and practical usage.

The microsporidian diversity catalogued so far has resulted in the development of several taxonomic groups, one of which is the Enterocytozoonida - a group of generalist 'ultimate opportunists', which infect many fished and aquacultured animals, as well as a broad suite of host taxa, including humans. In this study, we provide phylogenetic, ultrastructural, developmental, and pathological evidence for the creation of a new genus and species to hold a microsporidian parasite of the Jonah crab, Cancer borealis. Cancer borealis represents a species of commercial interest and has become the target of a recently developed fishery on the USA and Canadian Atlantic coast. This species was found to harbour a microsporidian parasite that develops in the cytoplasm of alpha and beta cells of the hepatopancreas. We retrieved a 937 bp fragment of the parasite SSU region, alongside developmental and ultrastructural data that suggests this species is ∼ 87 % similar to Parahepatospora carcini and develops in a similar manner in direct association with the host cell cytoplasm. The mature spores are ovoid in shape and measure 1.48 ± 0.15 µm (SD) in length and 1.00 ± 0.11 µm (SD) in width. Phylogenetically, the new parasite clades in the Enterocytozoonida on the same branch as P. carcini. We provide a new genus and species to hold the parasite: Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida) and explore the likelihood that this species may fit into the Hepatoporidae family.

Infestation with Varroa destructor compromises the survival of Apis mellifera colonies, especially during the winter season. In order to prevent colony losses due to the presence of Varroa mites, determination of the infestation level and the application of autumn treatment is highly recommended. However, autumn reinvasion by inter-apiary and intra-apiary horizontal transmission might threaten the control strategies. Drivers like the infestation level of untreated colonies, the presence of bee brood during late autumn and the colonies disposition within the apiary might explain at least partially the re-invasion rates of treated colonies during autumn and winter. A total of six apiaries with five colonies were distributed within a 30 km radius. Colonies were arranged within each apiary following a circular or a lineal disposition. Twenty-four of the thirty colonies were treated during late summer against V. destructor and one colony per apiary remained untreated. The phoretic Varroa infestation and the adult bee population, brood area and nutritional reserves in the colonies was estimated before and after treatment and then for the next four months (late autumn and winter season). In this study, a model for the V. destructor re-invasion dynamics during autumn and winter in honey bee colonies from a temperate climate was fitted. According to these results, the re-invasion probability is higher for lineal disposition of the colonies that also present a higher amount of brood availability during late autumn and winter. Beekeeping practical implications are discussed in the context of an Integrated Pest management approach.

White feces syndrome (WFS) is a commercially important disease in Penaeus vannamei (whiteleg shrimp) farming. The aetiology beyond the white or golden white midgut with mediocre growth performance producing a floating mass of white fecal strings in WFS-affected shrimp farms remains uncharted. To give WFS a perception of pathobiome, healthy P. vannamei shrimps were subjected to an enteric microsporidian Enterocytozoon hepatopenaei (EHP) infection along with Vibrio harveyi and V. alginolyticus in different combinations. Immune responses in haemolymph (total haemocyte count (THC), prophenoloxidase activity (proPO), respiratory burst activity (RBA), superoxide dismutase activity (SOD) and catalase activity (CAT)), plasma biochemical changes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and digestive enzymes activity (alpha-amylase (AMY), lipase (LIP) and protease (PRO)) were assessed in the challenged shrimps at 5, 10 and 15 days post-infection (dpi). The microbial interactions between the EHP and Vibrio spp. have led to the formation of WFS in the challenged shrimps. The histological sections of the hepatopancreas revealed the presence of EHP along with colonized bacterial masses, leading to the formation of aggregated transformed microvilli (ATM) structures and increased sloughing of lipid vacuoles into the tubule lumen. A significantly decreased THC and increased proPO levels, dysregulated antioxidant system, prominent hepatic damage, reduced energy metabolism and higher lipid production were the key records supporting that EHP-associated WFS in P. vannamei is due to the pathobiome.

Xenorhabdus, like other Gram-negative bacteria, possesses a Type 6 Secretion System (T6SS) which acts as a contact-dependent molecular syringe, delivering diverse proteins (effectors) directly into other cells. The number of T6SS loci encoded in Xenorhabdus genomes are variable both at the inter and intraspecific level. Some environmental isolates of Xenorhabdus bovienii, encode at least one T6SS locus while others possess two loci. Previous work conducted by our team demonstrated that X. bovienii [Jollieti strain SS-2004], which has two T6SSs (T6SS-1 and T6SS-2), hcp genes are required for biofilm formation. Additionally, while T6SS-1 hcp gene plays a role in the antibacterial competition, T6SS-2 hcp does not. In this study, we tested the hypothesis that vgrG genes are also involved in mutualistic and pathogenic interactions. For this purpose, targeted mutagenesis together with wet lab experiments including colonization, competition, biofilm, and virulence experiments, were carried out to assess the role of vgrG in the mutualistic and antagonistic interactions in the life cycle of XBJ. Our results revealed that vgrG genes are not required for biofilm formation but play a role in outcompeting other Xenorhabdus bacteria. Additionally, both vgrG and hcp genes are required to fully colonize the nematode host. We also demonstrated that hcp and vgrG genes in both T6SS clusters are needed to support the reproductive fitness of the nematodes. Overall, results from this study revealed that in X. bovieni jollieti strain, the twoT6SS clusters play an important role in the fitness of the nematodes in relation to colonization and reproduction. These results lay a foundation for further investigations on the functional significance of T6SSs in the mutualistic and pathogenic lifecycle of Xenorhabdus spp.

Tebuconazole (TEB) is a fungicide widely used in agriculture; however, its constant application has increased the emergence of resistant plant pathogenic fungal strains and reduced the effectiveness of fungi as biological control agents; for instance, the entomopathogenic and hyperparasitic fungus Akanthomyces lecanii, suitable for simultaneous biological control of insect pest and plant pathogenic fungi, is highly sensitive to fungicides. We carried out the induction of resistance to TEB in two wild type strains of A. lecanii by UV radiation and selective pressure in increasing fungicide gradients using a modified Microbial Evolution and Growth Arena (MEGA), to produce A. lecanii strains that can be used as biological control agent in the presence of tebuconazole. Nine UV-induced and three naturally adapted A. lecanii strains were resistant to TEB at the agriculturally recommended dose, and three irradiated strains were resistant to TEB concentration ten times higher; moreover, growth, sporulation rates, production of hydrolytic enzymes, and virulence against the hemipteran Coccus viridis, a major pest of coffee crops, were not affected in the TEB-resistant strains. These A. lecanii TEB-resistant strains would have a greater opportunity to develop and to establish themselves in fields where the fungicide is present and can be used in a combined biological-chemical strategy to improve insect and plant pathogenic fungal control in agriculture. Also, the selective pressure through modified MEGA plate methodology can be used for the adaptation of entomopathogenic filamentous fungi to withstand other chemical or abiotic stresses that limits its effectiveness for pest control.

Infection by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) has become a significant problem in the shrimp cultivation industry in Asian countries like Thailand, China, India, Vietnam, Indonesia, and Malaysia. The outbreak of this microsporidian parasite is predominantly related to the existence of macrofauna-carriers of EHP. However, information about potential macrofauna-carriers of EHP in rearing ponds is still limited. In this study, the screening of EHP in potential macrofauna-carriers was conducted in farming ponds of Penaeus vannamei in three states in Malaysia, namely Penang, Kedah, and Johor. A total of 82 macrofauna specimens (phyla: Arthropoda, Mollusca, and Chordata) were amplified through a polymerase chain reaction (PCR) assay targeting genes encoding spore wall proteins (SWP) of EHP. The PCR results showed an average prevalence of EHP (82.93%) from three phyla (Arthropoda, Mollusca and Chordata). The phylogenetic tree generated from the macrofauna sequences was revealed to be identical to the EHP-infected shrimp specimens from Malaysia (MW000458, MW000459, and MW000460), as well as those from India (KY674537), Thailand (MG015710), Vietnam (KY593132), and Indonesia (KY593133). These findings suggest that certain macrofauna species in shrimp ponds of P. vannamei are carriers of EHP spores and could be potential transmission vectors. This study provides preliminary information for the prevention of EHP infections that can be initiated at the pond stage by eradicating macrofauna species identified as potential vectors.

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is a disease of utmost concern in almost all shrimp growing countries. The pathogen was characterized by ultramicrography, histopathology and phylogenetic analysis of 18srDNA. A total of 183 biological samples were collected from all major shrimp growing states of the country.The histology technique could be used very well in identifying the site of infection and can aid in diagnosis of EHP. Wet mount and Ultramicrography were employed to observe the structure of spores. A single step PCR based method was developed for detecting the pathogen from variety of DNA samples including shrimp and non-shrimp sources.The developed PCR assay proved to be a robust and reliable technique to detect EHP in shrimps and environmental samples and for assessing the distribution of pathogen within geographical zones, thus aid in mitigating the disease. The PCR primers was also used to generate DIG labelled probe which was successful in binding to the EHP infected cells in HP of shrimp. The presence of pathogen was confirmed from many non-shrimp environmental samples suggests that they could act as reservoirs for recurrent infection in shrimp ponds. Proper control of these reservoirs will be the first step in recovering an EHP affected pond back to normal.

Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 ℃ for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 104 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.

The natural occurrence of entomopathogenic fungi (EPF) was investigated along the Ticino River (Ticino River Natural Park, Novara Province, Piedmont, Italy), at the center of the area of the first settlement of the invasive alien pest Popillia japonica. Using Zimmermann's "Galleria bait method", EPF were successfully isolated from 83 out of 155 soil samples from different habitats (perennial, cultivated, or uncultivated meadows, woodlands, and riverbanks). Sequencing of the 5' end of the Translation Elongation Factor 1 alfa (5'-TEF) region allowed the assignment of 94% of the isolates to Metarhizium spp., while 8% and 7% were assigned to Beauveria spp. and Paecilomyces spp., respectively. Four Metarhizium species were identified: Metarhizium robertsii was the most common one (61.5% of the isolates), followed by M. brunneum (24.4%), M. lepidiotae (9%), and M. guizhouense (5.1%). Microsatellite marker analysis of the Metarhizium isolates revealed the presence of 27 different genotypes, i.e., 10 genotypes among M. robertsii, 8 among M. brunneum, 5 among M. lepidiotae, and 4 among M. guizhouense. Metarhizium brunneum appeared to be associated with woodlands and more acid soils, while the other species showed no clear association with a particular habitat. Laboratory virulence tests against P. japonica 3rd instar larvae allowed the identification of one M. robertsii isolate that showed efficacy as high as 80.3%. The importance of this kind of study in the frame of eco-friendly microbiological control is discussed.

The origin of the sterility observed in ex-fissiparous freshwater planarians with hyperplasic ovaries has yet to be explained. To improve our understanding of this enigmatic phenomenon, immunofluorescence staining and confocal microscopy examination were used the assess autophagy, apoptosis, cytoskeleton, and epigenetics markers in the hyperplasic ovaries of ex-fissiparous individuals and the normal ovaries of sexual individuals. Immunofluorescence positivity for the autophagic marker microtubule-associated protein 1 light chain 3 (LC3) was significantly lower in the hyperplasic ovary than in the normal ovary. Compared with the normal ovary, the hyperplasic ovary exhibited significantly higher immunofluorescence positivity for the apoptotic marker caspase 3, suggesting that autophagy and apoptosis are closely associated in this pathogenicity. Furthermore, the level of global DNA (cytosine-5)-methyltransferase 3A (DNMT3) protein expression was significantly higher in the normal ovary than in the hyperplasic ovary, suggesting that DNA methylation is involved in the infertility phenomenon. The cytoskeleton marker actin also exhibited relatively higher immunofluorescence intensity in the normal ovary than in the hyperplasic ovary, consistent with previous findings on the role of cytoskeleton architecture in oocyte maturation. These results help improve our understanding of the causes of infertility in ex-fissiparous planarians with hyperplasic ovaries and provide new insights that will facilitate future studies on this mysterious pathogenicity.

Crayfishes are among the most widely introduced freshwater taxa and can have extensive ecological impacts. Knowledge of the parasites crayfish harbor is limited, yet co-invasion of parasites is a significant risk associated with invasions. In this study, we describe a novel microsporidium, Cambaraspora faxoni n. sp. (Glugeida: Tuzetiidae), from two crayfish hosts in the Midwest USA, Faxonius virilis and Faxonius rusticus. We also expand the known host range of Cambaraspora floridanus to include Procambarus spiculifer. Cambaraspora faxoni infects muscle and heart tissue of F. rusticus and develops within a sporophorous vesicle. The mature spore measures 3.22 ± 0.14 μm in length and 1.45 ± 0.13 μm in width, with 8-9 turns of the polar filament. SSU sequencing indicates the isolates from F. virilis and F. rusticus were identical (100%) and 93.49% similar to C. floridanus, supporting the erection of a new species within the Cambaraspora genus. The novel parasite was discovered within the native range of F. rusticus (Ohio, USA) and within a native congeneric (F. virilis) in the invasive range of F. rusticus (Wisconsin, USA). Faxonius virilis is invasive in other regions. This new parasite could have been introduced to Wisconsin with F. rusticus or it may be a generalist species with a broad distribution. In either case, this parasite infects two crayfish species that have been widely introduced to new drainages throughout North America and could have future effects on invasion dynamics or impacts.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) transmits the Gram-negative bacterium 'Candidatus Liberibacter asiaticus' that causes citrus greening disease. While chemical control has been the main management strategy for limiting D. citri, the widespread usage of chemical sprays has decreased the susceptibility of D. citri to most insecticides. Pesticidal proteins produced by the bacterium Bacillus thuringiensis (Bt) are active against a wide variety of insects and provide a more sustainable approach to insect control. Herein, we investigated the impact of 'Ca. L. asiaticus' infection of D. citri on the toxicity of two Bt proteins (Mpp51Aa1 and Cry1Ba1). Proteins were delivered to healthy and 'Ca. L. asiaticus'-infected D. citri via topical feeding application. The LC50 values of Mpp51Aa1 and Cry1Ba1 were calculated for both nymphs and adults. Additionally, we evaluated the effect of each protein on the survival probability and life span of healthy and 'Ca. L. asiaticus'-infected D. citri. The LC50 values indicated that adults and nymphs were more susceptible to Mpp51Aa1 than to Cry1Ba1 in both healthy and 'Ca. L. asiaticus'-infected D. citri. 'Ca. L. asiaticus'-infected adults and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than healthy insects, and nymphs were more susceptible to Mpp51Aa1 and Cry1Ba1 than adults. Moreover, we found that Mpp51Aa1 had a greater impact than Cry1Ba1 on the survival and lifespan of adults, and 'Ca. L. asiaticus'-infected insects were more affected by these pesticidal proteins than healthy adults. These results have important implications for the use of pesticidal proteins in D. citri management in Florida and elsewhere given the widespread presence of 'Ca. L. asiaticus' in the D. citri population. In this era of eco-friendly control strategies, Bt-derived pesticidal proteins provide a promising avenue to reducing the application of chemical insecticides for D. citri management.

Microsporidia are well known fungal pathogens of aquatic animals. However, the taxonomy of microsporidia is generally poorly resolved, which has consequently constrained our understanding of their pathology and epidemiology in marine animals. To date, microsporidia have been reported in both bivalves and gastropods, and microsporidia from mollusks have been classified in different genera. Despite ongoing work to better describe these genera, including detailed microscopic and ultrastructural images, so far we lack information on microsporidian phylogeny and pathogenicity of species within these genera. Here we investigate the microsporidian parasite Steinhausia mytilovum associated with the mussel, Mytilus galloprovincialis, in natural beds and farms along coast of southern Italy. A survey of M. galloprovincialis was conducted in 13 mussel farms and one natural bed between 2009 and 2020. We found the presence of S. mytilovum in 10 of the investigated farms, with a prevalence ranging between 14 and 100% of female mussels, depending on the population and season in which they were sampled. The parasite developed in the oocytes within a sporophorous vesicle (SV) where it produced 1-3 spores per cell, both in the cytoplasm and in the nucleus. Stenhausia mytilovum elicited an infiltrative (24.8%) or a strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective tissue, in some cases accompanied by gonadal atresia (24.8%), leading to loss of gonadal architecture. In 7% of cases no reaction was observed. Ultrastructural observations revealed a mitochondrial re-organization to interact with all the phases of parasite development; the mitochondria were arranged outside the parasitophorous vesicle (PV) or directly interacting with the spore inside vesicle. There are five taxonomic clades of microsporidians as identified by SSU ribosomal gene sequence data. Maximum likelihood analysis assigned S. mytilovum within the Clade IV, defined as the Class Terresporidia, with closest genetic relationship (83.6% identity) to an undetermined invertebrate ovarian microsporidian. The constant presence, prevalence, and severity of S. mytilovum in coastline populations of M. galloprovincialis populations in southern Italy may indirectly reflect immunocompetence at both individual and population levels.

Widespread native honey bee species in South and East Asia (Apis cerana, Apis dorsata and Apis florea) and the imported western honey bee (Apis mellifera) share habitats and potentially also share pathogens. Chief among the threats facing A. mellifera in Europe and North America is deformed wing virus (DWV), including its two principal genotypes: A and B (DWV-A and DWV-B respectively). Though DWV-A has been recorded in Asia's native Apis species, it is not known if DWV-B, or both DWV-A and DWV-B, are currently widespread in Asia and, if so, whether viral transmission is primarily intraspecific or interspecific. This study aims to fill these knowledge gaps by (i) determining the DWV genotype in four co-occurring Apis host species using qPCR and (ii) inferring viral transmission between them using nucleotide sequences of DWV from Apis host species collected at three independent localities in Northern Thailand. We found DWV-A and -B in all four Apis species, the exotic A. mellifera and the native A. cerana, A. dorsata and A. florea. That DWV-A sequences were identical across Apis species at the same locality, with a similar pattern for DWV-B sequences, suggests that DWV's epidemiology is largely driven by ongoing interspecific transmission (spillover) of DWV across co-occurring native and exotic Apis species. Both genotypes of DWV represent a serious threat to Asia's exotic and native honey bee species.

The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had ∼ 55% and ∼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in ∼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 μg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 μg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 μg/body weight) was administered than the group fed with a lower concentration of LPS (4 μg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

In Scandinavia, the bird cherry-oat aphid Rhopalosiphum padi overwinter as eggs on the bird cherry tree Prunus padus. Branches of P. padus were collected at the late February / early March from 17 locations in Norway over a three-year period. We found 3599 overwintering aphid eggs, 59.5% of which were dead. Further, a total of 879 overwintering fungus-killed cadavers were observed. These cadavers were found close to bud axils, where overwintering eggs were also usually attached. Cadavers were infected with either Zoophthora cf. aphidis or Entomophthora planchoniana. All the fungal-killed cadavers were filled with overwintering structures of Z. cf. aphidis (as resting spores) or E. planchoniana (as modified hyphal bodies). We found a significant negative correlation between eggs and cadavers per branch. However, both numbers of eggs and cadavers varied greatly between years and among tree locations. This is the first report of E. planchoniana overwintering in R. padi cadavers as modified hyphal bodies. We discuss whether P. padus may act as an inoculum reservoir for fungi infecting aphids in cereals in spring.

The life cycle of Echinocephalus pseudouncinatus has not been described in detail to date, as is also the case of other species in the genus. This study contributes to the information available about the life history of the genus, particularly E. pseudouncinatus, by (1) using molecular techniques (small subunit rRNA gene [SSU rRNA] sequences) to identify larvae of this species; (2) describing the most conspicuous morphological characteristics that distinguish the species by scanning electron microscopy; (3) describing histological characteristics of the internal anatomy of larvae and the lesions caused by them in the gonads of hosts; (4) reporting new host and reporting evidence of host specificity of E. pseudouncinatus between two species of edible pen shells coexisting in the northern Pacific of Mexico; and (5) analyzing the frequency of infested gonads by host sex, size, and gonadal development stage. The molecular analysis confirmed the morphological and histological diagnosis of E. pseudouncinatus larvae infesting gonads of Atrina maura and Pinna rugosa in Ojo de Liebre and San Ignacio lagoons. The morphological characterization of larvae provides internal details of the cephalic bulb (including rows of hooks and ballonets), neural chord, and cell types of the body wall. Histopathological findings such as degenerated and atretic gametes and the displacement of gonadal tissue in hosts infested with E. pseudouncinatus larvae are discussed in terms of temperature anomalies caused by the "Godzilla El Niño". The frequency of infestation increased with shell length and gonadal development in A. maura, with a higher infestation frequency in females versus males observed at both study sites. Our findings expand the list of hosts of E. pseudouncinatus, which was previously unknown as a parasite of P. rugosa. In P. rugosa, infestation frequencies are noticeably lower (<1 %) compared with those observed in A. maura (>30 %) at both study sites, suggesting host-specific requirements or preferences in E. pseudouncinatus larvae.

The new species Pandora cacopsyllae Eilenberg, Keller & Humber (Entomophthorales) is described. The fungus was found on infected pear psyllids Cacopsylla pyri (Hemiptera: Psyllidae) in a pear orchard in Zealand, Denmark. Morphological structures (conidia, rhizoids, cystidia) were described on the designated type host C. pyri. In addition, conidia from an in vitro culture were described. Pandora cacopsyllae differs from other Pandora species by a) C. pyri is the natural host; b) conidia are different from other Pandora species infecting Psylloidea; c) ITS differs from other Pandora species infecting Hemiptera. The fungus has a high potential for future use in biological control of Cacopsylla pest species as well as other psyllids.

Biological invasions may act as conduits for pathogen introduction. To determine which invasive non-native species pose the biggest threat, we must first determine the symbionts (pathogens, parasites, commensals, mutualists) they carry, via pathological surveys that can be conducted in multiple ways (i.e., molecular, pathological, and histological). Whole animal histopathology allows for the observation of pathogenic agents (virus to Metazoa), based on their pathological effect upon host tissue. Where the technique cannot accurately predict pathogen taxonomy, it does highlight pathogen groups of importance. This study provides a histopathological survey of Pontogammarus robustoides (invasive amphipod in Europe) as a baseline for symbiont groups that may translocate to other areas/hosts in future invasions. Pontogammarus robustoides (n = 1,141) collected throughout Poland (seven sites), were noted to include a total of 13 symbiotic groups: a putative gut epithelia virus (overall prevalence = 0.6%), a putative hepatopancreatic cytoplasmic virus (1.4%), a hepatopancreatic bacilliform virus (15.7%), systemic bacteria (0.7%), fouling ciliates (62.0%), gut gregarines (39.5%), hepatopancreatic gregarines (0.4%), haplosporidians (0.4%), muscle infecting microsporidians (6.4%), digeneans (3.5%), external rotifers (3.0%), an endoparasitic arthropod (putatively: Isopoda) (0.1%), and Gregarines with putative microsporidian infections (1.4%). Parasite assemblages partially differed across collection sites. Co-infection patterns revealed strong positive and negative associations between five parasites. Microsporidians were common across sites and could easily spread to other areas following the invasion of P. robustoides. By providing this initial histopathological survey, we hope to provide a concise list of symbiont groups for risk-assessment in the case of a novel invasion by this highly invasive amphipod.

The two-spotted lady beetle, Adalia bipunctata L., displays warning colouration that is reinforced by the production of adaline and adalinine. These alkaloids are thought to provide defense against predation throughout all life stages of A. bipunctata and may play a role in the insect immune system. Vairimorpha (Nosema) adaliae, a microsporidium described from A. bipunctata, has minimal effects on its host (delayed larval development) when reared under optimum conditions but stress factors are shown to affect the development of microsporidiosis. The objectives of this study were to determine the effects of V. adaliae on relative alkaloid content (adaline) during A. bipunctata development, and to evaluate the combined effects of physical stress and infection on adult beetles (relative alkaloid content and infection load). First-instar larvae were isolated from uninfected and V. adaliae-infected colonies. Eggs and first-instar larvae were immediately prepared for alkaloid analysis, whereas late-instar larvae, pupae and adults were systematically processed when each reached their designated developmental stage. Upon eclosion, a subsample of beetles was exposed to varying amounts of physical agitation: control (no shaking), alternate shaking (every other day), and daily shaking. Immediately following these stress trials, alkaloid samples were collected for analysis and spore loads were assessed. Overall, relative adaline proportions increased from egg to adult. Uninfected individuals had significantly higher relative proportions of adaline than did infected individuals during early development; however, adaline content was higher in infected A. bipunctata from the third-instar onwards, when compared to their uninfected counterparts. Following exposure to physical agitation on alternate days, uninfected adults had a significantly higher relative proportion of adaline than did infected adults. Interestingly, exposure to different levels of agitation had no significant effect on alkaloid production for either uninfected or infected beetles. Mean spore counts were significantly higher for adults that were exposed to daily shaking when compared to individuals from the control and alternate shaking groups. From a biological perspective, one would expect to observe differences in alkaloid production through coccinellid development, as each successive life stage faces different external pressures and risks. When infected with the microsporidium V. adaliae, however, adaline production was reduced during early development but increased significantly in late life stages.