Paranosema Locustae Research Articles

Transcriptomic analysis of the gonads of Locusta migratoria (Orthoptera: Acrididae) following infection with Paranosema locustae.

Paranosema locustae is an environmentally friendly parasitic predator with promising applications in locust control. In this study, transcriptome sequencing was conducted on gonadal tissues of Locusta migratoria males and females infected and uninfected with P. locustae at different developmental stages. A total of 18,635 differentially expressed genes (DEGs) were identified in female ovary tissue transcriptomes, with the highest number of DEGs observed at 1 day post-eclosion (7141). In male testis tissue transcriptomes, a total of 32,954 DEGs were identified, with the highest number observed at 9 days post-eclosion (11,245). Venn analysis revealed 25 common DEGs among female groups and 205 common DEGs among male groups. Gene ontology and Kyoto Encyclopaedia of Genes and Genome analyses indicated that DEGs were mainly enriched in basic metabolism such as amino acid metabolism, carbohydrate metabolism, lipid metabolism, and immune response processes. Protein-protein interaction analysis results indicated that L. migratoria regulates the expression of immune- and reproductive-related genes to meet the body's demands in different developmental stages after P. locustae infection. Immune- and reproductive-related genes in L. migratoria gonadal tissue were screened based on database annotation information and relevant literature. Genes such as Tsf, Hex1, Apolp-III, Serpin, Defense, Hsp70, Hsp90, JHBP, JHE, JHEH1, JHAMT, and VgR play important roles in the balance between immune response and reproduction in gonadal tissues. For transcriptome validation, Tsf, Hex1, and ApoLp-III were selected and verified by quantitative real-time polymerase chain reaction (qRT-PCR). Correlation analysis revealed that the qRT-PCR expression patterns were consistent with the RNA-Seq results. These findings contribute to further understanding the interaction mechanisms between locusts and P. locustae.

Alternative locust control with a dilutable linseed oil emulsion

AbstractLocust swarm control is still a main topic in agriculture and is often based on the extensive use of chemical agents that can be problematic for the environment and human health. Currently, only a few alternative control agents are available on the market, such as neem oil, the fungus Metarhizium sp., and the microsporidian Paranosema locustae. In this study, we improved the formulation of a botanical insecticide that is based on linseed oil and was found to be effective against the desert locust, Schistocerca gregaria. To make sure that linseed oil is most effective, we first tested several natural plant oils in a comparative laboratory study. Linseed oil performed best and resulted in 100% mortality within only 2 days after a single spray treatment. However, this linseed oil formulation contains three expensive essential oils, thus we further improved it to avoid costly essential oils. A much lower concentration of linseed oil was also found to be highly effective and killed adult S. gregaria within 3 days in an experiment performed under semifield conditions. Furthermore, a concentrated stock solution of this botanical insecticide was developed to increase stability and reduce shipping costs in areas where water for dilution is available. Mechanical treatment, applied during the preparation of this linseed oil emulsion, resulted in the formation of micro‐colloids, which prevent the separation of phases and enable 1:16 dilution with water containing bicarbonate. Interestingly, only 24 h after single spray treatment, locusts became immobile and most of them were unable to jump, which would make them easy prey for natural enemies. Later, insects died due to the hardening of the linseed oil emulsion. This novel linseed oil emulsion is harmless for humans, rather easy to produce, and remains stable over weeks in its concentrated form, which suggests that it is promising alternative agent for locust control.

The immune response of Locusta migratoria manilensis at different times of infection with Paranosema locustae.

Paranosema locustae is an entomopathogenic microsporidia with promising potential for controlling agricultural pests, including Locusta migratoria manilensis. However, it has the disadvantage of having a slow insecticidal rate, and how P. locustae infection impacts the host immune response is currently unknown. The present study investigated the effect of P. locustae on the natural immune response of L. migratoria and the activities of enzymes that protect against oxidative stress. Infection with P. locustae increased the hemocytes and nodulation number of L. migratoria at the initial stage of infection. The hemocyte-mediated modulation of immune response was also affected by a decrease in the number of hemocytes 12 days postinfection. Superoxide dismutase activity in locusts increased in the early stages of infection but decreased in the later stages, whereas the activities of peroxidase (POD) and catalase (CAT) showed opposite trends may be due to their different mechanisms of action. Furthermore, the transcription levels of mRNA of antimicrobial peptide-related genes and phenoloxidase activity in hemolymph in L. migratoria were suppressed within 15 days of P. locustae infection. Overall, our data suggest that P. locustae create a conducive environment for its own proliferation in the host by disrupting the immune defense against it. These findings provide useful information for the potential application of P. locustae as a biocontrol agent.

Molecular and biochemical changes in Locusta migratoria (Orthoptera: Acrididae) infected with Paranosema locustae.

Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen's life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.

Suppression of yolk formation, oviposition and egg quality of locust (Locusta migratoria manilensis) infected by Paranosema locustae.

Locusta migratoria manilensis is one of the most important agricultural pests in China. The locust has high fecundity and consumes large quantities of food, causing severe damage to diverse crops such as corn, sorghum, and rice. Immunity against pathogens and reproductive success are two important components of individual fitness, and many insects have a trade-off between reproduction and immunity when resources are limited, which may be an important target for pest control. In this study, adult females L. migratoria manilensis were treated with different concentrations (5 × 106 spores/mL or 2 × 107 spores/mL) of the entomopathogenic fungus Paranosema locustae. Effects of input to immunity on reproduction were studied by measuring feeding amount, enzyme activity, vitellogenin (Vg) and vitellogenin receptor (VgR) production, ovary development, and oviposition amount. When infected by P. locustae, feeding rate and phenol oxidase and lysozyme activities increased, mRNA expression of Vg and VgR genes decreased, and yolk deposition was blocked. Weight of ovaries decreased, with significant decreases in egg, length and weight.Thus, locusts used nutritive input required for reproduction to resist invasion by microsporidia. This leads to a decrease in expression of Vg and VgR genes inhibited ovarian development, and greatly decreased total fecundity. P. locustae at 2 × 107 spores/mL had a more obvious inhibitory effect on the ovarian development in migratory locusts. This study provides a detailed trade-off between reproduction and immune input of the female, which provides a reliable basis to find pest targets for biological control from those trade-off processes.

Nosema locustae (Protozoa, Microsporidia), a Biological Agent for Locust and Grasshopper Control

Effective locust and grasshopper control is crucial as locust invasions have seriously threatened crops and food security since ancient times. However, the preponderance of chemical insecticides, effective and widely used today, is increasingly criticized as a result of their adverse effects on human health and the environment. Alternative biological control methods are being actively sought to replace chemical pesticides. Nosema locustae (Synonyms: Paranosema locustae, Antonospora locustae), a protozoan pathogen of locusts and grasshoppers, was developed as a biological control agent as early as the 1980s. Subsequently, numerous studies have focused on its pathogenicity, host spectrum, mass production, epizootiology, applications, genomics, and molecular biology. Aspects of recent advances in N. locustae show that this entomopathogen plays a special role in locust and grasshopper management because it is safer, has a broad host spectrum of 144 orthopteran species, vertical transmission to offspring through eggs, long persistence in locust and grasshopper populations for more than 10 years, and is well adapted to various types of ecosystems in tropical and temperate regions. However, some limitations still need to be overcome for more efficient locust and grasshopper management in the future.

Infectivity of Paranosema locustae (Microsporidia) against gregarious-phase South American locust (Orthoptera) when treated en masse

En masse inoculations with Paranosema locustae, an intracellular parasite of adipose tissue of grasshoppers and locusts and the only microsporidium registered as a biocontrol agent, were conducted against crowded fourth-instar nymphs of the South American locust Schistocerca cancellata and the grasshoppers Dichroplus schulzi and Ronderosia bergii. Infection did not develop in the locust, but was highly prevalent in the two grasshopper species. We hypothesize that absolute absence of infection in S. cancellata may constitute a case of density-dependent prophylactic resistance, an elevation of the baseline immunity of an organism in order to cope with disease that is prevalent in species exhibiting phase polyphenism.

Effects of a combined infection with Paranosema locustae and Beauveria bassiana on Locusta migratoria and its gut microflora.

Even though Paranosema locustae is widely used in China as a biological agent for controlling grasshoppers, the mortality rate is initially quite low. This study sought to determine whether the simultaneous use of P. locustae and Beauveria bassiana would be a more effective control strategy. Additionally, changes in the intestinal microbial communities of migratory locusts infected with the two pathogens were analyzed to investigate the roles of gut microbes in pathogen-host interactions. The mortality rate of locusts inoculated with B. bassiana and P. locustae simultaneously was not significantly higher than expected, but the mortality rates of locusts inoculated with B. bassiana 3, 6, and 9 days after inoculation with P. locustae were significantly higher than if their effects were additive, indicating synergism. A MiSeq analysis found that Weissella was the most common bacterium, representing 41.48% and 51.62% of the total bacteria in the mid- and hindguts, respectively, and the bacterial declines were greatest during dual infections with B. bassiana and P. locustae. The appropriately timed combined application of P. locustae and B. bassiana was more effective against locusts than either treatment alone. Moreover, the combined inoculation of the two pathogens changed the gut microflora of locusts, indicating the potential relevancy of their synergistic effects on locust control.

Effects of Paranosema locustae (Microsporidia) on the development and morphological phase transformation of Locusta migratoria (Orthoptera: Acrididae) through modulation of the neurotransmitter taurine

Neurotransmitters are important in the maintenance of phase transformation of Locusta migratoria (Arthropoda: Orthoptera). Here, the effects of the entomopathogen Paranosema locustae on the neurotransmitter taurine in migratory locusts were studied using biochemical methods. After inoculation with P. locustae, the taurine content of infected locusts significantly declined, but F/C values (ratio between the length of hind femur and the width of the head of locust) increased significantly, compared to healthy locusts. Meanwhile, F/C values of infected locusts that were injected with 2 µg of taurine showed no significant differences from those of healthy locusts, demonstrating that supplemental taurine inhibited the changes in morphological phase caused by P. locustae. Paranosema locustae infection also caused longer developmental durations and lower body weights of locusts, but these changes were unaffected after injection with taurine. These results provided new insights into the mechanisms by which microsporidian parasites affected their locust hosts.

Biodiversity of beet webworm microsporidia in Eurasia

The beet webworm Loxostege sticticalis (LS) has eruptive type of population dynamics and high migratory activity. The LS outbreaks are therefore difficult to predict and the pest belongs to the category of highly dangerous pests. However, during periods of depressions this insect is not observed within the most of its range and is very susceptible to infection by pathogens, including various species of microsporidia, some of which are not specific parasites of the order Lepidoptera. The distribution of LS microsporidia in Eurasia is quite extensive. During the study period of LS from 2003 to 2019, we have found 6 species of microsporidia. The parasite list includes not only species known for Lepidoptera such as Nosema sp. and Vairimorpha thomsoni, but also Vairimorpha (Nosema) ceranae as the typical pathogen from Apis mellifera, as well as Endoreticulatus cf poecilimonae, a pathogen similar to Endoreticulatus poecilimonae from Poecilimon thoracicus. Moreover, two isolates from the genus Tubulinosema identified in LS belong to the group of parasites with a very wide host range, including humans. In laboratory experiments, LS proved high sensitivity to microsporidia N. pyrausta from Ostrinia nubilalis and N. tyriae from Tyria jacobaeae. Its susceptibility to Paranosema locustae from Locusta migratoria has also been discovered.

Dynamics of Aboveground Natural Enemies of Grasshoppers, and Biodiversity after Application of Paranosema locustae in Rangeland

Substantial harm to ecosystems from the use of chemical pesticides has led to an increasing interest in the use of biopesticides to control grasshoppers in rangelands, including China. One such potential biopesticide for control of grasshoppers is the fungus Paranosema locustae. In this study, the dynamics of aboveground natural enemies of grasshoppers and arthropod diversity 0–9 years after application of P. locustae were investigated in rangeland in Qinghai Plateau, China. We found that the number of species and of individuals of aboveground natural enemies increased by 17–250% and 40–126%, respectively, after spraying P. locustae, and that the main natural enemies showed three peaks after treatment. The conventional indices of species diversity (H’) and evenness (J’) increased by 11–267% and 13–171%, respectively, after treatment with P. locustae. The results showed the positive effects of P. locustae on aboveground natural enemies and biodiversity in an arthropod community in Chinese rangeland. Paranosema locustae is thought to be a safe biological control agent for grasshopper management in Northwestern China.

Efficacy of Two Entomopathogenic Fungi, Metarhizium brunneum, Strain F52 Alone and Combined with Paranosema locustae against the Migratory Grasshopper, Melanoplus sanguinipes, under Laboratory and Greenhouse Conditions.

Grasshopper outbreaks cause significant damage to crops and grasslands in US. Chemical control is widely used to suppress these pests but it reduces environmental quality. Biological control of insect pests is an alternative way to reduce the use of chemical insecticides. In this context, two entomopathogenic fungi, Metarhizium brunneum strain F52 and Paranosema locustae were evaluated as control agents for the pest migratory grasshopper Melanoplus sanguinipes under laboratory and greenhouse conditions. Third-instar grasshoppers, reared in the laboratory, were exposed up to fourteen days to wheat bran treated with different concentrations of each of the fungi alone or the two pathogens combined. In the greenhouse, nymphs were placed individually in cages where they were able to increase their body temperatures by basking in the sun in an attempt to inhibit the fungal infection, and treated with each pathogen alone or in combination. Mortality was recorded daily and presence of fungal outgrowth in cadavers was confirmed by recording fungal mycosis for two weeks’ post-treatment (PT). For combination treatment, the nature of the pathogen interaction (synergistic, additive, or antagonistic effects) was also determined. In laboratory conditions, all treatments except P. locustae alone resulted in grasshopper mortality. The application of the pathogen combinations caused 75% and 77%, mortality for lower and higher concentrations, respectively than each of the pathogens alone. We infer a synergistic effect occurred between the two agents. In greenhouse conditions, the highest mortalities were recorded in combination fungal treatments with a M. brunneum dose (60% mortality) and with a combination of the two pathogens in which M. brunneum was applied at high rate (50%) two weeks after application. This latter combination also exhibited a synergistic effect. Exposure to the P. locustae treatment did not lead to mortality until day 14 PT. We infer that these pathogens are promising for developing a biopesticide formulation for rangeland pest grasshopper management.

Hexokinase as a versatile molecular genetic marker for Microsporidia.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3'-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

Increase of Albinistic Hosts Caused by Gut Parasites Promotes Self-Transmission.

Paranosema locustae is a gut parasite that has been applied widely in the control of grasshoppers in many parts of the world. Usually, P. locustae is transmitted horizontally via passive modes under natural conditions but in the current study, a positive transmission strategy of P. locustae was demonstrated. First, infection by P. locustae resulted in the cuticula of infected Locusta migratoria nymphs to become lighter in color: normally only a small proportion of locusts are pale with most either being partly or mostly black; but locusts infected with P. locustae became pale. And it was found that the change to pale occurred even among uninfected black and partly black nymphs reared with infected locusts. The eumelanin of the thorax and abdomen of infected individuals decreased significantly, as did the level of dopamine. In addition, there was a decrease in phenol oxidase activity and the expression of henna and pale, which are involved in the synthesis of cuticle melanin, decreased. What is the ecological significance of this increase in light-colored hosts caused by P. locustae? We discovered that light-colored locusts were more susceptible to the microsporidian pathogen than dark-colored individuals were, because of their weaker melanization. Phenol oxidase activity in pale locusts was lower than that of black locusts, but the serpin expression level of pale locusts was higher than that of black individuals. When examined for infection, it was found that initially uninfected nymphs had picked up P. locustae infections indicating that infections are readily passed from one pale locust to another. The infection rate of healthy locusts reared with light-colored locusts infected with P. locustae was 100% which was more than with black-colored ones. The increase in albinistic locusts clearly promoted the prevalence of P. locustae in the total population. In conclusion, these results elucidated a new strategy of positive self-transmission in P. locustae.Importance:Mother Nature always grants wisdom to her creatures and feeds them carefully. This wisdom is particularly apparent in the relationships between two interacting species. In this study, our team focused on the interaction between L. migratoria and P. locustae. In a previous study, it was found that L. migratoria isolate infected individuals, reducing avoiding the spread of P. locustae, in a previous study. The solitary, pale individuals infected by P. locustae were left behind as locust groups marched ahead, leading to a kind of behavioral immunity in the insects. Here, we reported that P. locustae promotes pigmentation loss in L. migratoria, causing a larger proportion of light-colored individuals, and these lighter individuals which possessed weaker immunity against pathogens. This strategy is advantageous to P. locustae, as it promotes its propagation and spread. These extraordinary abilities of L. migratoria and P. locustae have accumulated over millennia of years of interaction.

The creation of a recombinant single-chain antibody against α/β-hydrolase of microsporidium Paranosema locustae and immunolocalization of the enzyme in an infected host cell

Microsporidia are a group of widespread fungi-related obligate intracellular parasites. Direct contact of most microsporidia with the cytoplasm of an infected host cell entails possible secretion of various proteins from the parasite that allows control physiological processes of the host. Earlier, by means of polyclonal antibodies against α/β-hydrolase of microsporidium Paranosema locustae, the secretion of large amounts of the enzyme into the cytoplasm of fat body cells of infected migratory locust Locusta migratoria was demonstrated. However, yeast fungi Pichia pastoris did not recognize this enzyme as a secretory one during its heterologous expression. In the present study, a library of recombinant single-chain antibodies (scFv fragments) against proteins of the infected fat body of locust was constructed. The use of the phage display technology enabled choosing a miniantibody that specifically recognized the studied enzyme. Immunoblotting and immunolabeling of frozen sections of locust fat body with the selected scFv fragment confirmed the fact of secretion of P. locustae α/β-hydrolase (as two forms of different size) into the infected host cell. Prospects of using the selected scFv fragment for further studies of the secretion mechanism of the parasite’s protein and its role in host–parasite interactions are discussed.

A Novel Spore Wall Protein from Antonospora locustae (Microsporidia: Nosematidae) Contributes to Sporulation

Microsporidia are obligate intracellular parasites, existing in a wide variety of animal hosts. Here, we reported Aloc SWP2, a novel protein identified from the spore wall of Antonospora locustae (formerly, Nosema locustae, and synonym, Paranosema locustae), containing four cysteines that are conserved among the homologues of several Microspodian pathogens in insects and mammals. Aloc SWP2 was detected in the wall of mature spores via indirect immunofluorescence assay. In addition, immunocytochemistry localization experiments showed that the protein was observed in the wall of sporoblasts, sporonts, and meronts during sporulation within the host body, also in the wall of mature spores. AlocSWP2 was not detected in the fat body of infected locust until the 9th day after inoculating spores via RT‐PCR experiments. Furthermore, the survival percentage of infected locusts injected with dsRNA of AlocSWP2 on the 15th, 16th, and 17th days after inoculation with microsporidian were significantly higher than those of infected locusts without dsRNA treatment. Conversely, the amount of spores in locusts infected with A. locustae after treated with RNAi AlocSWP2 was significantly lower than those of infected locusts without RNAi of this gene. This novel spore wall protein from A. locustae may be involved in sporulation, thus contributing to host mortality.

The development of the microsporidium Paranosema (Nosema) locustae for grasshopper control: John Henry’s innovation with worldwide lasting impacts

The development of the microsporidium Paranosema (Nosema) locustae for grasshopper control: John Henry’s innovation with worldwide lasting impacts

Heterologous expression of Paranosema (Antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei

Paranosema (Nosema, Antonospora) locustae is the only microsporidium produced as a commercial product for biological control. Molecular mechanisms of the effects of this pathogen and other invertebrate microsporidia on host cells remain uncharacterized. Previously, we immunolocalized P. locustae hexokinase in nuclei of Locusta migratoria infected adipocytes. Here, the microsporidian protein was expressed in the yeast Pichia pastoris and in lepidopteran Sf9 cells. During heterologous expression, P. locustae hexokinase was accumulated in the nuclei of insect cells but not in yeast cell nuclei. This confirms nuclear localization of hexokinase secreted by microsporidia into infected host cells and suggests convenient model for its further study.

The mechanism for microsporidian parasite suppression of the hindgut bacteria of the migratory locust Locusta migratoria manilensis.

Locusts aggregate into bands of nymphs and swarms of adults that can pose a major threat to crop. Previous studies have shown that infection by the microsporidian parasite Paranosema locustae prevents locust aggregation behavior and we show that gut bacteria, which produce components of locust aggregation pheromones, are substantially reduced in locusts infected with P. locustae. We found that P. locustae could reduce the diversity, abundance and community composition of Locusta migratoria’s gut bacteria. The parasite infection was also shown to interrupt the peroxidase activity of locust hindgut. Genome-wide expression analysis showed that the parasite infection suppressed peroxidase mRNA relative expression of locust hindgut, but had no effects on attacin expression and superoxide dismutase at 16 d post-inoculation with 20,000 P. locustae spores. Our findings reveal the mechanisms by which P. locustae impairs bacterial diversity and community structure of Locusta migratoria’s gut bacteria.

Effect of Paranosema locustae (Microsporidia) on the behavioural phases of Locusta migratoria (Orthoptera: Acrididae) in the laboratory

The ability of parasites to modify the behaviour of their hosts is a wide spread phenomenon, but the effects of microsporidian parasites on locust behaviour remain unexplored. Here the frequencies of directional changes (ND) and jumping (NJ) per minute of gregarious locusts infected with 2000 spores of the microsporidian parasite Paranosema locustae were significantly different from those of untreated locusts 10 and 16 days after infection, being similar to values for solitary nymphs. In contrast, the behaviour of locusts inoculated with the lower doses of 200 spores/locust was sometimes like that of solitary nymphs. At other times, behaviour was intermediate between solitary and gregarious, i.e. transitional. The rearing density did not affect the turning and jumping behaviour of infected locusts, and their behaviours were similar to those of solitary locusts at 10–16 days after infection. Our study demonstrates that infection with P. locustae may lead gregarious locusts to change some of their behaviour to that typical of solitary locusts.