Production of Escovopsis conidia and the potential use of this parasitic fungus as a biological control agent of leaf-cutting ant fungus gardens.

This study was carried out to investigate the use of different substrates for the production of Escovopsis conidia and verify the virulence of four different isolates cultured on four types of substrates using a novel bioassay. Escovopsis isolates were molecularly identified, based on Internal Transcribed Spacer (ITS) nucleotide sequences. To evaluate conidial production, suspensions (1 × 106 conidia mL-1) of each Escovopsis isolate were inoculated onto four substrates (parboiled rice, white rice, rolled oats, and corn grits). After 14 days, conidial yields were assessed. The virulence of each isolate cultured on the four substrates was tested against Leucoagaricus fungus garden fragments, by directly applying 500 µL of each conidial suspension (1 × 107 conidia mL-1), and the development of the parasite was monitored daily until it completely colonized the fungus garden. It was observed that rolled oats were the best substrate for conidial production, with a yield of 1.7 × 107 to 2.0 × 108 conidia mL-1. Furthermore, isolate AT-01 produced the highest number of conidia when compared with the other isolates. Regardless of the substrate used to produce AT-01 conidia, this isolate completely colonized the fungus garden 6 days post inoculation (dpi), followed by AT-02, AC-01, and AC-2. High levels of both conidial production and virulence against the leaf-cutting ant fungus garden were observed here.

Differential parasitism by four species of phorid flies when attacking three worker castes of the leaf-cutting ant Atta laevigata (Smith, 1858).

Certain species of parasitic flies belonging to the Phoridae are known to attack Atta spp. workers foraging along trails, near nest openings used by the ants to supply the colony with plant material, and in the areas where the ants are actively cutting plant material. However, there have been no previous studies of phorid parasitism of non-foraging worker ants, for example excavators and soldiers. Excavators can be found on the surface around specialized nest openings, carrying and dumping soil on characteristic mounds. Soldiers can be found on the trails protecting foragers or guarding the different types of nest openings. The current study was performed to investigate the differential parasitism rates of Atta laevigata (Smith, 1858) worker castes by four species of phorids. Ants of all castes on trails and at nest entrances were collect from 18 mature colonies in the field. A total of 21,254 ants were collected from trails and 14,649 collected from the mounds of loose soil near nest openings. The captured workers were maintained under controlled laboratory conditions to evaluate the rate of parasitism. Of the ants collected from trails, 1,112 (5.23%) were found to have been parasitized, of which 1,102 were foragers and only 10 were soldiers. Of the ants collected from the soil mounds near the nest openings, only 27 (0.18%) were found to have been parasitized, of those 25 were excavators and 2 were soldiers. When evaluating parasitism of ants on the trails, 46.2% were attacked by Apocephalus attophilus Borgmeier, 1928, 22.6% by Myrmosicarius grandicornis Borgmeier, 1928, 16.6% by Eibesfeldtphora erthali (Brown, 2001) and 14.6% by Apocephalus vicosae Disney, 2000. Only two species of phorid, M. grandicornis and E. erthali, were observed parasitizing excavators, whilst only E. erthali parasitized soldiers. This is the first time that Atta spp. excavators and soldiers have been shown to be parasitized by phorids. The low rates of parasitism and specificity of certain phorid species for excavators and soldiers is discussed in relation to the behavioral interactions of hosts and their parasitoids, as well as the relationship between host and parasitoid size.

Biological performance and oviposition preference of tomato pinworm Tuta absoluta when offered a range of Solanaceous host plants.

The tomato pinworm Tuta absoluta (Lepidoptera: Gelechuidae) is native to South America and has now become the main tomato pest in Europe, Africa and Asia. The wide range of host plants attacked by this pest has been reported as one of the main reasons for the success of this important insect species. However, the information currently available on the biological performance of T. absoluta on Solanaceae has been obtained from a limited number of host species. The Solanaceae family is composed of thousands of species, many of which are potential hosts for T. absoluta. Our results showed that the highest oviposition rates occurred on cultivated tomato plants, potato and wild tomato. The lowest rates occurred on "gilo", "jurubeba", green pepper and pepper. The highest survival rates of the immature stages occurred on potato and the lowest on pepper, green pepper and "jurubeba". Female fertility, following infestation of the different plant species, was highest for insects that developed on tomato or potato and the lowest rates were seen on American black nightshade. The net reproductive rate and the intrinsic growth rate were highest on potato and tomato. Cluster analysis grouped tomato and potato as highly susceptible to attack, American black nightshade, juá, eggplant, gilo and wild tomato as moderately susceptible, whilst pepper, green pepper and jurubeba were categorized as resistant to T. absoluta. These results clearly demonstrate that the choice of solanaceous host plant species has a direct impact on the fitness parameters of the tomato pinworm as well as survival potential, dispersion and establishment at new sites. These results are important for the planning of integrated pest management strategies.

Neem oil increases the persistence of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: The entomopathogenic fungus Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions. METHODS: Neem was mixed with M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis. RESULTS: Laboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation. CONCLUSIONS: A combination of the entomopathogenic fungus M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.

Phaseolin ingestion affects vesicular traffic causing oxidative stress in the midgut of Callosobruchus maculatus larvae.

It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.

Natural mortality factors of tomato leafminer Tuta absoluta in open-field tomato crops in South America.

BACKGROUND: Little importance has been given to the role of natural mortality factors (biotic and abiotic) in the regulation of tomato leafminer Tuta absoluta (Lepidoptera: Gelechiidae) populations. The present study determined the action of mortality factors on T. absoluta populations infesting cultivated tomato crops. Eighty ecological life tables for T. absoluta in field cultivated tomato plants were constructed and analyzed. RESULTS: Total T. absoluta mortality was 99.08%, with 38.76% mortality during the egg phase, 57.20% in the larva phase and 3.12% in the pupal phase. The main mortality factors during the egg stage were predation, parasitism and egg inviability. In the larval stage, the main mortality factors were predation, parasitism, entomopathogenic agents and physiological disorders. In the pupal stage, the main mortality factor was predation. The larvae of the third and fourth instar were more susceptible to the action of mortality factors and the predatory wasp, Protonectarina sylveirae, was the main insect predator of these larvae. CONCLUSIONS: The T. absoluta population is regulated under field conditions by the action of natural enemies of the larvae. The predatory wasp P. sylveirae is very important in the regulation of T. absoluta populations in open-field tomato crops in Brazil. © 2018 Society of Chemical Industry.

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Improving the delivery and efficiency of fungus-impregnated cloths for control of adult Aedes aegypti using a synthetic attractive lure.

BACKGROUND: Entomopathogenic fungi are highly promising agents for controlling Aedes aegypti mosquitoes. Deploying fungus-impregnated black cloths in PET traps efficiently reduced Ae. aegypti female survival rates under intra-domicile conditions. With the aim of further increasing the effectiveness of the traps, the addition of attractive lures to fungus-impregnated traps was evaluated. METHODS: Black cloths were suspended inside 2 l plastic bottles called "PET traps". These traps were placed in rooms simulating human residences. The first experiments evaluated the attraction of mosquitoes to PET traps with black cloths covered in adhesive film with and without synthetic lures (AtrAedes™). Traps were left in the test rooms for either 24 or 48 h. The attractiveness of the lures over time was also evaluated. The efficiency of PET traps with fungus-impregnated black cloths associated with lures was compared to that of traps without lures. RESULTS: The highest percentage of captured mosquitoes (31 and 66%) were observed in PET traps with black cloths covered in adhesive film + attractive lure maintained in test rooms for 24 h and 48 h, respectively. Black cloths covered in adhesive film captured 17 or 36% of the mosquitoes at 24 h and 48 h, respectively. The attractiveness of the lures fell gradually over time, capturing 37% after 5 days on the bench and 22% of the mosquitoes after 30 days exposure to ambient conditions. Associating attractive synthetic lures with black cloths impregnated with M. anisopliae placed in test rooms for 120 h reduced mean survival to 32%, whilst black cloths impregnated with M. anisopliae without lures resulted in a 48% survival rate. Using Beauveria bassiana in the traps resulted in a 52% reduction in mosquito survival, whilst combining Beauveria and AtrAedes resulted in a 36% survival rate. PET traps impregnated with fungus + AtrAedes resulted in similar reductions in survival when left in the rooms for 24, 48, 72 or 120 h. CONCLUSIONS: AtrAedes increased attractiveness of PET traps with black cloths under intra-domicile conditions and when associated with M. anisopliae or B. bassiana, significantly reduced Aedes survival. This strategy will reduce the number of PET traps necessary per household.

Effects of dietary replacement of fishmeal by mealworm meal on muscle quality of farmed shrimp Litopenaeus vannamei.

This study investigated the muscle quality of the shrimp Litopenaeus vannamei fed on a diet containing different proportions of mealworm meal (MW) (0, 25, 50, 75 and 100%) as a substitute for fishmeal, which is the normal diet used in shrimp commercial production. The proximate composition, fatty acid profile, colour and texture of the shrimps were evaluated. Moisture, protein, and ash content of shrimp muscle were not significantly altered when fishmeal was replaced by MW (p>0.05). However, the replacement resulted in a linear increase in lipid content (p<0.05). The fatty acid composition of the experimental diets directly mirrored the fatty acid composition of shrimp muscle. The absence of long-chain polyunsaturated fatty acids in MW composition resulted in a linear decrease in eicosapentaenoic and docosahexaenoic fatty acids in shrimp muscle with increasing levels of MW in the diet. The n-3/n-6 ratio ranged from 0.50 to 0.67. Colour and firmness were unchanged between the treatments. Although the use of MW as a fishmeal substitute in L. vannamei diets has affected the lipid and fatty acid composition of shrimp muscle, from a human nutritional perspective, the lipid content of the shrimps is considered low and the n-3/n-6 ratio remained within the human dietary requirements. Therefore the use of a mealworm diet for shrimp production is a viable alternative to increasingly expensive fishmeal based diets.

Receptor mediated endocytosis of vicilin in Callosobruchus maculatus (Coleoptera: Chrysomelidae) larval midgut epithelial cells.

The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin.

Metarhizium brunneum Blastospore Pathogenesis in Aedes aegypti Larvae: Attack on Several Fronts Accelerates Mortality.

Aedes aegypti is the vector of a wide range of diseases (e.g. yellow fever, dengue, Chikungunya and Zika) which impact on over half the world's population. Entomopathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have been found to be highly efficacious in killing mosquito larvae but only now are the underlying mechanisms for pathogenesis being elucidated. Recently it was shown that conidia of M. anisopliae caused stress induced mortality in Ae. aegypti larvae, a different mode of pathogenicity to that normally seen in terrestrial hosts. Blastospores constitute a different form of inoculum produced by this fungus when cultured in liquid media and although blastospores are generally considered to be more virulent than conidia no evidence has been presented to explain why. In our study, using a range of biochemical, molecular and microscopy methods, the infection process of Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores was investigated. It appears that the blastospores, unlike conidia, readily adhere to and penetrate mosquito larval cuticle. The blastospores are readily ingested by the larvae but unlike the conidia are able infect the insect through the gut and rapidly invade the haemocoel. The fact that pathogenicity related genes were upregulated in blastospores exposed to larvae prior to invasion, suggests the fungus was detecting host derived cues. Similarly, immune and defence genes were upregulated in the host prior to infection suggesting mosquitoes were also able to detect pathogen-derived cues. The hydrophilic blastospores produce copious mucilage, which probably facilitates adhesion to the host but do not appear to depend on production of Pr1, a cuticle degrading subtilisin protease, for penetration since protease inhibitors did not significantly alter blastospore virulence. The fact the blastospores have multiple routes of entry (cuticle and gut) may explain why this form of the inoculum killed Ae. aegypti larvae in a relatively short time (12-24hrs), significantly quicker than when larvae were exposed to conidia. This study shows that selecting the appropriate form of inoculum is important for efficacious control of disease vectors such as Ae. aegypti.

Neem oil increases the efficiency of the entomopathogenic fungus Metarhizium anisopliae for the control of Aedes aegypti (Diptera: Culicidae) larvae.

BACKGROUND: Entomopathogenic fungi are potential candidates for use in integrated vector management and many isolates are compatible with synthetic and natural insecticides. Neem oil was tested separately and in combination with the entomopathogenic fungus Metarhizium anisopliae against larvae of the dengue vector Aedes aegypti. Our aim was to increase the effectiveness of the fungus for the control of larval mosquito populations. METHODS: Commercially available neem oil was used at concentrations ranging from 0.0001 to 1%. Larval survival rates were monitored over a 7 day period following exposure to neem. The virulence of the fungus M. anisopliae was confirmed using five conidial concentrations (1 × 10(5) to 1 × 10(9) conidia mL(-1)) and survival monitored over 7 days. Two concentrations of fungal conidia were then tested together with neem (0.001%). Survival curve comparisons were carried out using the Log-rank test and end-point survival rates were compared using one-way ANOVA. RESULTS: 1% neem was toxic to A. aegypti larvae reducing survival to 18% with S50 of 2 days. Neem had no effect on conidial germination or fungal vegetative growth in vitro. Larval survival rates were reduced to 24% (S50 = 3 days) when using 1 × 10(9) conidia mL(-1). Using 1 × 10(8) conidia mL(-1), 30% survival (S50 = 3 days) was observed. We tested a "sub-lethal" neem concentration (0.001%) together with these concentrations of conidia. For combinations of neem + fungus, the survival rates were significantly lower than the survival rates seen for fungus alone or for neem alone. Using a combination of 1 × 10(7) conidia mL(-1) + neem (0.001%), the survival rates were 36%, whereas exposure to the fungus alone resulted in 74% survival and exposure to neem alone resulted in 78% survival. When using 1 × 10(8) conidia mL(-1), the survival curves were modified, with a combination of the fungus + neem resulting in 12% survival, whilst the fungus alone at this concentration also significantly reduced survival rates (28%). CONCLUSIONS: The use of adjuvants is an important strategy for maintaining/increasing fungal virulence and/or shelf-life. The addition of neem to conidial suspensions improved virulence, significantly reducing larval survival times and percentages.

Spatial distribution of digestive proteinases in the midgut of the Pacific white shrimp (Litopenaeus vannamei) indicates the existence of endo-ectoperitrophic circulation in Crustacea.

The effect of dietary protein concentration on the spatial distribution of digestive proteinases in the shrimp Litopenaeus vannamei indicates the existence of endo-ectoperitrophic enzyme circulation in this species. Samples recovered from the midgut gland tissues, stomach contents, three different portions of the midgut and feces were used for quantitative and qualitative analyses of the composition and distribution of the digestive proteinases. Animals were divided into three different groups: (1) animals (controls) fed with a commercial 35% protein diet, (2) animals fed with a commercial diet supplemented with ovalbumin to a final protein concentration of 60%; (3) animals fed with an 80% protein diet. Quantitative determinations using different substrates and zymograms showed that increasing protein concentration in the diet alters the distribution of proteinases along the digestive tract. Composition of proteinases in the midgut gland, stomach contents, midgut sections and feces were similar, but not identical. Chymotrypsin and trypsin paralogues were identified in all enzyme sources in a concentration gradient along the midgut in the control shrimp, the expected distribution supporting the existence of a recycling mechanism. The occurrence of a peritrophic membrane in other Decapoda suggests that endo-ectoperitrophic circulation of digestive enzymes and nutrients may also occur in other crustaceans and also extends beyond the Insecta.

Monitoring persistence of the entomopathogenic fungus Metarhizium anisopliae under simulated field conditions with the aim of controlling adult Aedes aegypti (Diptera: Culicidae).

BACKGROUND: Entomopathogenic fungi are potential candidates for use in integrated vector management, with recent emphasis aimed at developing adult mosquito control methods. Here we investigated the persistence of the fungus Metarhizium anisopliae when tested against female A. aegypti under field conditions. METHODS: Black cotton cloths impregnated with M. anisopliae conidia, formulated in vegetable oil + isoparaffin, were maintained on a covered veranda for up to 30 days. At specific times, pieces of the cloths were removed, placed in Tween 80 and the resuspended conidia were sprayed directly onto mosquitoes. The persistence of conidia impregnated on black cloths using three different carriers was evaluated in test rooms. Fifty mosquitoes were released into each room and after a 5 day period, the surviving insects were captured. Another 50 insects were then released into each room. The capacity of the fungus at reducing mosquito survival was evaluated over a total of 35 days. RESULTS: Conidia extracted from cloths maintained on the veranda for 2 to 18 days remained virulent, with 28 to 60% mosquito survival observed. Mosquito survival following exposure to fungus impregnated cloths showed that fungus + Tween caused similar reductions to that of fungus + vegetable oil. Mosquitoes exposed to the formulation fungus + vegetable oil had survival rates of 36% over the first 5 days of the experiment. Following the release of the second cohort of mosquitoes (6-11days), survival increased to 50%. The survival of the 12-17 day cohort (78%) was statistically equal to that of the controls (84%). Formulation of the fungus in vegetable oil + isoparaffin increased the persistence of the fungus, with the 18-23 day cohort (64% survival) still showing statistical differences to that of the controls (87% survival). CONCLUSIONS: The potential of entomopathogenic fungi for the control of adult A. aegypti was confirmed under field conditions. Vegetable oil + isoparaffin formulations of M. anisopliae significantly increased the effectiveness of the fungus, thus reducing the need for frequent changes of black cloths in residences. Our future aim is to obtain effective control of mosquito populations, with cloths only needing to being replaced once a month.

Digestion in adult females of the leaf-footed bug Leptoglossus zonatus (Hemiptera: Coreidae) with emphasis on the glycoside hydrolases α-amylase, α-galactosidase, and α-glucosidase.

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α-glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane-bound isoforms, being more abundant as a membrane-bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion-exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α-amylases and α-galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α-amylases acting on maize seed starch granules.

Variant vicilins from a resistant Vigna unguiculata lineage (IT81D-1053) accumulate inside Callosobruchus maculatus larval midgut epithelium.

It has been demonstrated that variant vicilins are the main resistance factor of cowpea seeds (Vigna unguiculata) against attack by the cowpea beetle Callosobruchus maculatus. There is evidence that the toxic properties of these storage proteins may be related to their interaction with glycoproteins and other microvillar membrane constituents along the digestive tract of the larvae. New findings have shown that following interaction with the microvilli, the vicilins are absorbed across the intestinal epithelium and thus reach the internal environment of the larvae. In the present paper we studied the insecticidal activity of the variant vicilins purified from a resistant cowpea variety (IT81D-1053). Bioassays showed that the seeds of this genotype affected larval growth, causing developmental retardation and 100% mortality. By feeding C. maculatus larvae on susceptible and IT81D-1053 derived vicilins (FITC labelled or unlabelled), followed by fluorescence and immunogold cytolocalization, we were able to demonstrate that both susceptible and variant forms are internalized in the midgut cells and migrate inside vesicular structures from the apex to the basal portion of the enterocytes. However, when larvae were fed with the labelled vicilins for 24h and then returned to a control diet, the concentration of the variant form remained relatively high, suggesting that variant vicilins are not removed from the cells at the same rate as the non-variant vicilins. We suggest that the toxic effects of variant vicilins on midgut cells involve the binding of these proteins to the cell surface followed by internalization and interference with the normal physiology of the enterocytes, thereby affecting larval development in vivo.

Testing fungus impregnated cloths for the control of adult Aedes aegypti under natural conditions.

BACKGROUND: Entomopathogenic fungi could be useful tools for reducing populations of the dengue mosquito Aedes aegypti. Here the efficiency of fungus (Metarhizium anisopliae) impregnated cloths (with and without imidacloprid [IMI]) was evaluated against adult A. aegypti in simulated human dwellings. Behaviour of mosquitoes in the presence of black cloths was also investigated. FINDINGS: When mosquitoes were released into the test rooms, the lowest survival rates (38%) were seen when five black cloths impregnated with conidia of ESALQ 818 + 10 ppm IMI were fixed under tables and chairs. This result was significantly lower than the survival rate recorded when cloths were impregnated with ESALQ 818 alone (44%) or ESALQ 818 + 0.1 ppm IMI (43%). Blood fed A. aegypti had lower landing frequencies on black cloths than sucrose fed insects during the first 24 h following feeding, which may have been due to reduced flight activity. Few mosquitoes (4-5%) were observed to land on the cloths during the hours of darkness. The landing pattern of sucrose-fed mosquitoes on non-treated and fungus-treated cloths was similar. CONCLUSION: The synergism between M. anisopliae and IMI significantly reduced Aedes survival in simulated field conditions. The use of fungus impregnated cloths is a promising point source application method for the control of adult A. aegypti.

Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.

The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-ß-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, α-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

Symbiotic bacteria on the cuticle of the leaf-cutting ant Acromyrmex subterraneus subterraneus protect workers from attack by entomopathogenic fungi.

Although only discovered in 1999, the symbiotic filamentous actinobacteria present on the integument of certain species of leaf-cutting ants have been the subject of intense research. These bacteria have been shown to specifically suppress fungal garden parasites by secretion of antibiotics. However, more recently, a wider role for these bacteria has been suggested from research revealing their generalist anti-fungal activity. Here we show, for the first time, evidence for a role of these bacteria in the defence of young worker ants against a fungal entomopathogen. Experimental removal of the bacterial bio-film using an antibiotic resulted in a significant increase in susceptibility of worker ants to infection by the entomopathogenic fungus Metarhizium anisopliae. This is the first direct evidence for the advantage of maintaining a bacterial bio-film on the cuticle as a defensive strategy of the ants themselves and not exclusively for protection of the fungus garden.

Susceptibility of adult female Aedes aegypti (Diptera: Culicidae) to the entomopathogenic fungus Metarhizium anisopliae is modified following blood feeding.

BACKGROUND: The mosquito Aedes aegypti, vector of dengue fever, is a target for control by entomopathogenic fungi. Recent studies by our group have shown the susceptibility of adult A. aegypti to fungal infection by Metarhizium anisopliae. This fungus is currently being tested under field conditions. However, it is unknown whether blood-fed A. aegypti females are equally susceptible to infection by entomopathogenic fungi as sucrose fed females. Insect populations will be composed of females in a range of nutritional states. The fungus should be equally efficient at reducing survival of insects that rest on fungus impregnated surfaces following a blood meal as those coming into contact with fungi before host feeding. This could be an important factor when considering the behavior of A. aegypti females that can blood feed on multiple hosts over a short time period. METHODS: Female A. aegypti of the Rockefeller strain and a wild strain were infected with two isolates of the entomopathogenic fungus M. anisopliae (LPP 133 and ESALQ 818) using an indirect contact bioassay at different times following blood feeding. Survival rates were monitored on a daily basis and one-way analysis of variance combined with Duncan's post-hoc test or Log-rank survival curve analysis were used for statistical comparisons of susceptibility to infection. RESULTS: Blood feeding rapidly reduced susceptibility to infection, determined by the difference in survival rates and survival curves, when females were exposed to either of the two M. anisopliae isolates. Following a time lag which probably coincided with digestion of the blood meal (96-120 h post-feeding), host susceptibility to infection returned to pre-blood fed (sucrose fed) levels. CONCLUSIONS: Reduced susceptibility of A. aegypti to fungi following a blood meal is of concern. Furthermore, engorged females seeking out intra-domicile resting places post-blood feeding, would be predicted to rest for prolonged periods on fungus impregnated black cloths, thus optimizing infection rates. It should be remembered that lowered susceptibility was only a temporary phenomenon and this may not necessarily occur when mosquitoes are infected with other fungal isolates. These results may have implications for field testing of entomopathogenic fungi by our group and further studies should be carried out to better understand the insect-fungus interaction.