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.     

Effect of Paranosema (Nosema) locustae (Microsporidia) on morphological phase transformation of Locusta migratoria manilensis (Orthoptera: Acrididae)

Field and laboratory studies demonstrated that Paranosema (Nosema) locustae had significant effects on the morphological phase transformation of Locusta migratoria manilensis (Meyen 1835). In the field, spraying P. locustae on gregarious locusts caused a substantial population reduction by 16 days after treatment, with most of the surviving locusts being phase solitaria. However, the effects of P. locustae on locust phase transformation began before direct mortality had caused a substantial reduction in locust density: locust numbers were still high at day 10, but locusts had already transformed to phase transiens. Laboratory assays showed that while a low dose of P. locustae had no effect on phase transformation, at a higher dose of 1×10⁵ spores/mL, locusts had F/C ratios that were significantly (P<0.05) more solitaria than untreated locusts, with locusts having ratios that were either phase solitaria or on the solitaria side of phase transiens. In a second laboratory experiment that analysed the effects of locust density on phase transformation by P. locustae, there was no obvious effect of density on female locusts 10 days later as all were solitaria at all locust densities. At day 16, female locusts were transiens at higher densities, but were solitaria at 4/cage. With males there were lesser effects. These results provide new evidence for P. locustae having sub-lethal effects on locust phase transformation at a wide range of locust densities.     

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 AlocSWP2, 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. AlocSWP2 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.     

Differential susceptibility of two locust pests to the microsporidium Paranosema (Antonospora) locustae at suboptimal rearing temperature

Paranosema (Antonospora) locustae (Canning) is a microsporidium with an extensive host range including >100 reported insect hosts from the order Orthoptera. The susceptibility of two species of locusts (Orthoptera: Acrididae) – the migratory locust, Locusta migratoria subsp. migratorioides (Fairmaire & Reiche), and the desert locust, Schistocerca gregaria Forsskål – to P. locustae was compared under laboratory conditions at a decreased temperature of 27 °C to enhance susceptibility to infection. Locusta migratoria was found highly vulnerable as infection percentages exceeded 70% at 10⁴, 10⁵, and 10⁶ spores per insect, and mortality increased with increasing dosage. Conversely, P. locustae spores were not found in S. gregaria throughout the experiment. Only at 10⁷ spores per insect, as many as 20% of S. gregaria were infected. This suggests that the desert locust is resistant to P. locustae infection, as opposed to the migratory locust. The laboratory models of these parasite–host systems may be useful for elucidating mechanisms of insect immunity to microsporidia.     

Reduction of consumption by grasshoppers (Orthoptera: Acrididae) infected with Nosema locustae canning (Microsporida: Nosematidae)

The effect of ingestion of Nosema locustae Canning spores on feeding by grasshoppers was measured in simultaneous laboratory and field experiments. After 21 days, fourth-instar Melanoplus sanguinipes (F.) nymphs, administered spores at the rates of 0, 2.0 × 10⁴, 2.0 × 10⁵, and 2.0 × 10⁶ per grasshopper, showed dry matter consumption of 102, 87, 64, and 26 mg in 48 hr, respectively. Rate of inoculation was a significant factor in suppression of feeding after correction for the effects of developmental stage, sex, and body weight. The quantity of dry matter consumed decreased linearly with increasing rate of spore ingestion. Experiments on 50 caged 1-m² plots on pasture grass yielded similar trends in per capita consumption independent of the effects of mortality. Field consumption per integrated grasshopper-day was 108, 77, 31, and 27 mg dry wt at the four inoculation rates, over 20 days.     

Bacterial catalase in the microsporidian Nosema locustae: implications for microsporidian metabolism and genome evolution

Microsporidia constitute a group of extremely specialized intracellular parasites that infect virtually all animals. They are highly derived, reduced fungi that lack several features typical of other eukaryotes, including canonical mitochondria, flagella, and peroxisomes. Consistent with the absence of peroxisomes in microsporidia, the recently completed genome of the microsporidian Encephalitozoon cuniculi lacks a gene for catalase, the major enzymatic marker for the organelle. We show, however, that the genome of the microsporidian Nosema locustae, in contrast to that of E. cuniculi, encodes a group II large-subunit catalase. Surprisingly, phylogenetic analyses indicate that the N. locustae catalase is not specifically related to fungal homologs, as one would expect, but is instead closely related to proteobacterial sequences. This finding indicates that the N. locustae catalase is derived by lateral gene transfer from a bacterium. The catalase gene is adjacent to a large region of the genome that appears to be far less compact than is typical of microsporidian genomes, a characteristic which may make this region more amenable to the insertion of foreign genes. The N. locustae catalase gene is expressed in spores, and the protein is detectable by Western blotting. This type of catalase is a particularly robust enzyme that has been shown to function in dormant cells, indicating that the N. locustae catalase may play some functional role in the spore. There is no evidence that the N. locustae catalase functions in a cryptic peroxisome.     

New microsporidia parasitizing bark lice (Insecta: Psocoptera)

Two species of bark lice, Xanthocaecilius sommermanae Mockford and Polypsocus corruptus Hagen, collected in a canopy Malaise trap placed in Great Smoky Mountains National Park as part of a survey of the park’s fauna, were found to be infected with microsporidia. Diagnosis was originally based on light microscopy, and was confirmed by PCR amplification and electron microscopy. This is the first record of microsporidia infection in the insect order Psocoptera. Four morphological spore types corresponded to four original SSUrDNA sequences (Genbank accession no. FJ865221-24), suggesting infection with four microsporidia species. Two of those species were examined by electron microscopy. We describe here one new genus and two new species based on morphological and sequence data: Antonospora psocopterae sp. n. with elongated diplokaryotic spores, 4.4 ± 0.05 × 1.9 ± 0.03 μm and Mockfordia xanthocaeciliae gen. n. sp. n. with ovocylindrical monokaryotic spores, 2.5 ± 0.10 × 1.4 ± 0.02 μm. A. psocopterae displayed high sequence (95%) and structural similarity with Antonospora scoticae, fell within a well supported dichotomy with A. scoticae inside the Antonospora-Paranosema clade in phylogenetic analyses by NJ, PS and ML. M. xanthocaeciliae did not exhibit much sequence or structural similarity with any of known microsporidia species, except Encephalitozoon spp. M. xanthocaeciliae fell within one clade with Encephalitozoon spp. in phylogenies and shared with encephalitozoons structural resemblance and about 80% of SSUrDNA sequence identity. The other two species were not described and provisionally were placed to the collective genus Microsporidium as Microsporidium sp. 1 and Microsporidium sp. 4 from bark lice because of insufficient morphological data. The finding that samples fixed and stored for months in propylene glycol (“antifreeze”) are good enough for DNA sequence analysis and can be used for morphological analyses (if no better fixation alternatives are available), is promising for future surveys for microsporidia.     

Molecular and ultrastructural characterization of Andreanna caspii n. gen., n. sp. (Microsporida: Amblyosporidae), a parasite of Ochlerotatus caspius (Diptera: Culicidae)

A new genus and species of microsporidia, Andreanna caspii n. gen., n. sp. is described from the mosquito, Ochlerotatus caspius (Pallas) based on ultrastructural morphology, developmental characteristics, and comparative sequence analyses of the small subunit (SSU) ribosomal DNA (rDNA). Parasite development is confined to fat body tissue and infected larvae appear swollen with dull white masses within the thorax and abdomen. Meronts have diplokaryotic nuclei and are delineated by a simple plasmalemma contiguous with the host cell cytoplasm. Merogony occurs by synchronous binary division followed by cytokinesis. Diplokaryotic sporonts undergo meiosis and synchronous nuclear division forming sporogonial plasmodia with two, four and eight nuclei enclosed within a persistent sporophorous vesicle. Cytokinesis of sporogonial plasmodia results in the formation of eight uninucleate spores. The episporontal space of early sporonts is filled with a homogeneous accumulation of electron dense granular inclusions and ovoid vesicles of various dimensions, transforming into an interwoven matrix during the initial phase of sporogenesis. Spores are oval, uninucleate and measure 4.8 ± 0.3 × 3.1 ± 0.4 μm (fixed). The spore wall is 260 μm thick with an irregular exospore consisting of two layers (150-170 μm) and a thinner endospore (90-100 μm). The anchoring disk is well developed and is contiguous with a lamellar polaroplast that occupies the anterior third of the spore and possess more narrow lamellae on the posterior end. The polar filament is gradually tapered and arranged in a single row consisting of six coils ranging from 180 to 150 μm in diameter. The posterior vacuole (posterosome) is moderately sized and filled with a matrix of moderate electron density. Phylogenetic analysis of SSU rDNA from A. caspii and 30 other species of microsporidia including 11 genera parasitic in mosquitoes using maximum parsimony, neighbor joining and maximum likelihood methods showed A. caspii to be a sister group to the clade containing all of the Amblyospora species, including Culicospora, Edhazardia and Intrapredatorus, as well as Culicosporella and Hyalinocysta thus providing strong support for establishment of Andreanna as a separate genus.     

Susceptibility of immature stages of the locusts Schistocerca gregaria and Locusta migratoria migratorioides to the microsporidium Johenrea locustae and effects of infection on feeding and fertility in the laboratory

Second instar nymphs of African migratory locust, Locusta migratoria migratorioides, and desert locust, Schistocerca gregaria, were tested for their susceptibility to the microsporidium pathogen Johenrea locustae (Lange et al. 1996 Lange, C.E., Becnel, J.J., Razafindratiana, E., Przybyszewski, J. and Razafindrafara, H. 1996. Johenrea locustae n.g., n.sp. (Microspora: Glugeidae): A Pathogen of Migratory Locusts (Ortrhoptera: Acrididae: Oedipodinae) from Madagascar. Journal of Invertebrate Pathology, 68: 28–40.  [Google Scholar], Journal of Invertebrate Pathology, 68, 28) in the laboratory. Spores of J. locustae were produced from live L.m. migratorioides, a conspecific to L. migratoria capito. Locusta m. migratorioides and S. gregaria were exposed to wheat seedlings sprayed with 20 mL of three concentrations (10⁶, 10⁷ and 10⁸ spores mL^?1). Both second-instar nymphs of L.m. migratorioides and S. gregaria were susceptible to J. locustae infection at the three concentrations. There was no effect of concentration of the microsporidium on mortalities of S. gregaria (92–98%) 19 days postinfection. Adjusted mortality in L.m. migratorioides at 19 days post-exposure was 24, 43 and 80% at the corresponding treatment concentrations. The effect of infection on fecundity was tested on both L.m. migratorioides and S. gregaria. Spinach leaf discs were treated with different concentrations (0, 10⁴, 10⁵ and 10⁶ spores mL^?1) of J. locustae and presented to female insects for 24 h. Female L.m. migratorioides surviving infection as nymphs laid significantly fewer egg pods than untreated controls at all levels of exposure. The number of eggs per female was also significantly lower in treated lots than in the controls. Higher spore concentrations also adversely affected egg hatching rate. The effect of J. locustae infection on feeding was tested on S. gregaria. There was a significant decrease in food intake among S. gregaria nymphs treated only at the high concentration (10⁶ spores mL^?1).     

Field observations of the effect of Metarhizium anisopliae var. acridum on the saxaul locust,Dericorys albidula Serville (Orthoptera: Dericorythidae)

Dericorys albidula Serville (Orthoptera: Dericorythidae) is a major pest of Haloxylon ammodendron and other saxaul plant species in the Qom province, Iran. Using fungal insecticides can be an alternative method for chemical insecticides. Effect of insecticide fungi, Metarhizium anisopliae var. acridum, on the various nymphs of D. albidula was studied in the field through 2005 and 2006. Fixed concentrations of fungi (10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹³ spore mL^?1) were prepared as gasoline formulation and were sprayed on the locusts on H. ammodendron trees, and mortality percentage was recorded 15 days after treatment. The results showed that various concentrations significantly affected on the second, third, fourth and fifth nymphal instars of D. albidula compared to control in 2006, although this effect was lower in 2005 on nymphs. Mortality of the highest concentration (10¹³ spore mL^?1) was higher (17.6–24%) than other concentrations in all tests, but these values were not notable. The results of this study showed that using M. anisopliae var. acridum diluted in gasoline can be effective on nymphal instars of locust, D. albidula, in two continuous years.     

Infection of Locusta migratoria with entomopoxviruses from Arphia conspersa and Melanoplus sanguinipes grasshoppers

Entomopoxvirus (EPV) occlusion bodies isolated from Arphia conspersa and Melanoplus sanguinipes grasshoppers were fed to 3rd and 4th instar Locusta migratoria nymphs. Locus mortality induced by A. conspersa EPV was first detected 18 days after addition of virus to the diet, and reached a level of approximately 68% of the colony population by 60 days after virus inoculation. In a similar population of L. migratoria nymphs, mortality induced by M. sanguinipes virus reached 90% 60 days after virus inoculation. Entomopoxvirus was isolated from M. sanguinipes EPV infected locust nymphs and the viral DNA was cleaved with several restriction endonucleases. The DNA fragment patterns obtained after agarose gel electrophoresis were compared with the fragment patterns from the original sample of M. sanguinipes EPV DNA cleaved with the same restriction endonucleases. No differences in the cleavage patterns were detected between the two virus DNA samples. Virus structural proteins of M. sanguinipes EPV purified from infected locust nymphs were compared by polyacrylamide gel electrophoresis with virus proteins isolated from the original sample of M. sanguinipes EPV. A total of six different virus protein bands were detected between the two poxvirus preparations.     

New case of long-term persistence of Paranosema locustae (Microsporidia) in melanopline grasshoppers (Orthoptera: Acrididae: Melanoplinae) of Argentina

We report an additional case of long-term persistence of Paranosema locustae in grasshoppers of Argentina. The pathogen was introduced from North America on rangeland at Loncopué, Neuquén province. Microsporidia were not detected in pre-introduction samples whereas infected grasshoppers were found 11 years after introduction. Affected grasshoppers were the melanoplines Dichroplus elongatus, Dichroplus maculipennis, and Scotussa lemniscata, some of them with high spore loads. The case highlights the ability of P. locustae to recycle in local grasshopper communities by parasitizing susceptible species other than the natural hosts.     

Laboratory assessment of the potential of Paranosema locustae to control immature stages of Schistocerca gregaria and Oedaleus senegalensis and vertical transmission of the pathogen in host populations

We tested the effects of Paranosema locustae spores in wheat bran formulation on the immature stages of Schistocerca gregaria and Oedaleus senegalensis under laboratory conditions. Younger instars were the most sensitive to the pathogen. While 100% infection was recorded in younger instar nymphs, older instars were less sensitive, with 16–27% of the inoculated nymphs remaining uninfected at the end of the experiment. Mortality of each instar increased with increased spore concentration. Immature survival time was significantly reduced by the pathogen and none of the nymphs inoculated as first, second, and third instar nymphs developed to adulthood (6–30% and 55–74% of nymphs inoculated as fourth and fifth instar, respectively). Sublethal effects such as delayed host growth, reduced host size, and abnormal wing and leg development (37% of emerging adults) were noted. Almost half the infected adults showed morphological abnormalities at emergence. Moreover, infection in S. gregaria and O. senegalensis by P. locustae did not affect female oviposition. However, 60% of S. gregaria and 52% of O. senegalensis progeny clearly showed infection by P. locustae with infection intensity of 1.08±0.27×10¹ and 1.19±0.32×10² spores/nymph, respectively. In view of the mortality rates, immature survival, host growth, and abnormal development in the P. locustae treatments, and the high prevalence of the pathogen in offspring from infected parents, it can be expected that the reduction in the impact of the two acridid species in the field will be considerable.     

Secretion of Antonospora (Paranosema) locustae Proteins into Infected Cells Suggests an Active Role of Microsporidia in the Control of Host Programs and Metabolic Processes

Molecular tools of the intracellular protozoan pathogens Apicomplexa and Kinetoplastida for manipulation of host cell machinery have been the focus of investigation for approximately two decades. Microsporidia, fungi-related microorganisms forming another large group of obligate intracellular parasites, are characterized by development in direct contact with host cytoplasm (the majority of species), strong minimization of cell machinery, and acquisition of unique transporters to exploit host metabolic system. All the aforementioned features are suggestive of the ability of microsporidia to modify host metabolic and regulatory pathways. Seven proteins of the microsporidium Antonospora (Paranosema) locustae with predicted signal peptides but without transmembrane domains were overexpressed in Escherichia coli. Western-blot analysis with antibodies against recombinant products showed secretion of parasite proteins from different functional categories into the infected host cell. Secretion of parasite hexokinase and α/β-hydrolase was confirmed by immunofluorescence microscopy. In addition, this method showed specific accumulation of A. locustae hexokinase in host nuclei. Expression of hexokinase, trehalase, and two leucine-rich repeat proteins without any exogenous signal peptide led to their secretion in the yeast Pichia pastoris. In contrast, α/β-hydrolase was not found in the culture medium, though a significant amount of this enzyme accumulated in the yeast membrane fraction. These results suggest that microsporidia possess a broad set of enzymes and regulatory proteins secreted into infected cells to control host metabolic processes and molecular programs.     

Bioassay method for assessing the virulence of Beauveria bassiana against tarnished plant bug, Lygus lineolaris (Hem., Miridae)

A standard bioassay method for assessing the pathogenicity of Beauveria bassiana (Balsamo) Vuillemin (GHA strain) against second instar tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hem., Miridae) was developed. Several types of inoculation methods, assay containers and incubation times were tested. Our goal was to minimize control mortality and maximize treatment mortality. Five inoculation methods (immersing broccoli florets or bean pods, spraying broccoli florets or bean pods, and immersing insects) and four types of plastic containers (114‐, 171‐, 228‐ and 455‐ml) were tested. Immersing insects directly in a fungal suspension was the most effective inoculation method, which resulted in a treatment mortality of 70–81.3% at a concentration of 1 × 10⁷ conidia/ml. The 114‐ml plastic container was the most suitable assay container when 10 tarnished plant bug nymphs were treated together, resulting in a control mortality of only 6% 12 days after treatment. Within the first 6 days after treatment, 71.1% of the insects were killed, compared with a total mortality of 81.3% after 12 days. Nymphs infected with the test fungus changed colour from green to black. Mycelial outgrowth and sporulation on the cadavers demonstrated that most nymphs died of fungal infection. A total of 61.1 and 80.5% of the cadavers showed signs of mycelial outgrowth 9 days after death among those that were surface sterilized and those that were not, respectively.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

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.     

Infectivity,viability and effects of Paranosema locustae (Microsporidia) on juveniles of Dichroplus maculipennis (Orthoptera: Acrididae: Melanoplinae) under laboratory conditions

Infectivity and effects on host of a long-term stored aqueous suspension of Paranosema locustae on juveniles of Dichroplus maculipennis, a pest grasshopper in parts of the Pampas and Patagonia, were evaluated. Infections developed in 90–97.8% of treated individuals. Mortality increased with time, reaching highest values at 30–40 days post-inoculation (79.5–100%). Infected nymphs showed significantly slower development.     

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min⁻¹. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.     

Effects of Metarhizium anisopliae on Meccus pallidipennis (Hemiptera: Reduviidae) over different types of wall surfaces

The entomopathogenic fungus Metarhizium anisopliae is virulent for the insect triatomine Meccus pallidipennis. To evaluate the functionality of a fungal formulation (vegetable oil and emulsifiers) of this fungus, virulence was assayed by insect mortality on the pronotum of third instar nymphs (N3) M. pallidipennis in laboratory conditions and ST₅₀ was calculated. Mortality was evaluated directly: 100%, 97.33% and 98.66% mortalities were caused by formulation, emulsified formulation and fungal conidia, respectively, at day 8 of insect infection. Another bioassay was carried out in simulated external conditions (peridomicility) using red and gray brick walls, a stone fence and mountain soil (experimental units). These simulated conditions were infected with 10?ml of a 1?×?10⁹?conidia/ml emulsified formulation by means of a manual sprinkler prior to the placement of the nymphs. Ten N3 M. pallidipennis were deposited in each experimental unit and insect mortality was monitored every 12?h for 22 days. Each treatment was replicated four times. With the red brick wall, a mortality of 90% at day 22 and a ST₅₀ of 15 days were obtained on N3 M. pallidipennis; with the gray brick wall, 100% mortality and a ST₅₀ of 13 days; and with the stone fence, 88.33% mortality and a ST₅₀ of 21 days. The results obtained in this research work indicate that the formulation with conidia of the M. anisopliae strain EH-473/4 may be auxiliary in the development of strategies for the control of Chagas disease insect transmitters such as M. pallidipennis.