Azadirachtin-based insecticide impairs testis morphology and spermatogenesis of the southern armyworm Spodoptera eridania (Lepidoptera: Noctuidae).

BACKGROUND: In the search for alternative tools for integrated pest management, azadirachtin, a botanical insecticide, has been used with the most promising activity against Spodoptera spp., but the mechanism of cytotoxicity on reproductive organs remains unclear. Spodoptera eridania (Stoll, 1782) is a polyphagous pest with great economic importance that has become an important target to elucidate the action of azadirachtin on the reproductive organs of insect pests, helping to understand the deleterious effects caused by its exposure. This study evaluated the effects of chronic exposure to azadirachtin on the morphology and ultrastructure of S. eridania larval testes as well as larval development. RESULTS: Azadirachtin exposure (6 or 18 mg a.i. L-1 ) caused a progressive increase in cumulative mortality and reduced gain in body mass after 5 days. Testicular structure indicated a reduction in their size with internal morphological changes such as spermatogonia, spermatogonial, spermatocytes and spermatid cysts in degeneration. The occurrence of cell death in germ and somatic cells was evidenced by the TUNEL technique. Electron microscopy revealed changes in cystic cells, such as cytoplasmic membrane rupture and cytoplasmic vacuolization. Chromatin compaction, changes in the rough endoplasmic reticulum and Golgi complex cisternae were observed in germ cells. Apoptotic bodies occurred between germ cell cysts. CONCLUSION: Azadirachtin damaged the testes of S. eridania larvae, and these changes compromised spermatogenesis and consequently the development of the reproductive potential of this specimen, making azadirachtin a promising botanical insecticide for application in integrated pest management programs. © 2022 Society of Chemical Industry.

Bioactivity of Bacillus thuringiensis (Bacillales: Bacillaceae) on Diatraea saccharalis (Lepidoptera: Crambidae) eggs.

BACKGROUND: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that synthesizes specific protein toxins, which can be exploited for control of various insect pests, including Diatraea saccharalis, a lepidopteran that severely damages sugarcane crops. Although studies have described the effects of Bt in the larval phases of D. saccharalis, few have examined its effect on insect eggs. Herein, we studied the entomopathogenic potential of Bacillus thuringiensis serovar Aizawai GC-91 (Bta) during D. saccharalis embryo development with the aim of understanding the entomopathogenic mechanism and developing new biological control techniques for target insects. RESULTS: Bta concentrations of 5, 10 and 20 g L-1 demonstrated the strongest bioactivity, reducing D. saccharalis egg viability by 28.69%, 33.91% and 34.98%, respectively. The lethal concentrations (LCs) were estimated as: LC50 = 28.07 g L-1 (CI 95% = 1.89-2.38) and LC90 = 65.36 g L-1 (CI 95% = 4.19-5.26). Alterations in egg coloration, melanization and granule accumulation were observed at 24 h, persisting until 144 h. The embryo digestive systems were severely damaged, including narrowing of the intestinal lumen, vesiculations and degenerated cells, causing embryonic death. CONCLUSION: The toxicity caused by Bta in D. saccharalis embryos demonstrated its potential as a biological control agent and as a sustainable alternative for integrated management of D. saccharalis infestation. © 2020 Society of Chemical Industry.

Developmental changes in larvae of the oriental latrine fly, Chrysomya megacephala, exposed to deltamethrin.

The Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) has a worldwide distribution and is considered of medical and veterinary importance. Its synanthropic behavior combined with the visitation of decaying organic matter allows it to mechanically transmit pathogens to human and animals. Pyrethroids are widely used pesticides, effective in low doses against a broad spectrum of insects. Our objective was to evaluate the action of K-Othrine® on the post-embryonic development of C. megacephala. First instar larvae were exposed to different concentrations of K-Othrine® (3.75 × 10-5 g a.i/mL (low), 7.5 × 10-5 g a.i/mL (intermediate low), 1.125 × 10-4 g a.i/mL (intermediate), 1.5 × 10-4 g a.i/mL (intermediate high), and 2.5 × 10-4 g a.i/mL (high)) mixed into artificial diets and a control with no pesticide. We measured weight, time of development, sex ratio, larval and pupal mortality, and esterase patterns. The mean weight values at intermediate low and intermediate concentrations were significantly lower compared with the control; additionally, the development time was delayed compared with the control. The larval and pupal mortality differed among the treatments. The larvae had six esterase regions, which altered its relative activity at 48 h and 72 h after insecticide ingestion. Further studies must investigate the effects of different insecticides on the post-embryonic development of C. megacephala.

Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungifrom yellowpassion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitroantagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichumas the most frequent genus and minor occurrence of Curvulariaendophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichumsp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plantsand other treatments. Despite many fungiof Colletotrichumgenus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichumendophytes living in asymptomatic relationship. By the way,we come across a Colletotrichumsp. endophyte able to antagonize a Colletotrichumsp. pathogen

Action of Bacillus thuringiensis (Bacillales: Bacillaceae) in the midgut of the sugarcane borer Diatraea saccharalis (Fabricius, 1794) (Lepidoptera: Crambidae).

The relevance of Bacillus thuringiensis in pest control strategies have been increasing in recent decades. This entomopathogenic microorganism is considered safe, and its action is highly selective to target organisms. Its toxic effect is attributed to the toxins synthesized during sporulation. The nature of the produced toxins depends on the bacterial variety. Some varieties of B. thuringiensis can synthesize from one to eight distinct toxins, which have specific effects on different orders of susceptible insects. The sugarcane borer, Diatraea saccharalis, is a holometabolous lepidopteran that causes severe damage to sugar cane cultures during its larval phase. Therefore, to evaluate B. thuringiensis serovar Aizawai GC-91 efficiency and its effects on the midgut of first instar D. saccharalis larvae, we reared the insects with contaminated artificial diet for 144 h. Larvae mortality during this period revealed that 5, 10, and 20 g/L concentrations were the most efficient, resulting in 100% mortality of treated larvae, with LC50 and LC90 estimated at 0.307 and 1.330 g/L, respectively. Furthermore, histological and ultrastructural analyses revealed damage to midgut cells. Within the first 24 h of treatment, the midgut of infected insects presented peritrophic membrane degeneration, cytoplasmic vacuolization, and cellular hypertrophy; spherites and calcium granules were also observed. At 48 h of treatment, the digestive system had collapsed, with microvilli degeneration, basement membrane and muscular fiber bundles disruption, and cellular lysis. These results confirm the toxicity of the B. thuringiensis serovar Aizawai GC-91 on D. saccharalis larvae and its potential use as a biocontroller against this pest species.

Bioprospection of immature salivary glands of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae).

Larval therapy (LT) comprises the application of sterile Calliphoridae larvae for wound debridement, disinfection, and healing in humans and animals. Larval digestion plays a key role in LT, where the salivary glands and gut produce and secrete proteolytic and antimicrobial substances. The objective of this work was to bioprospect the salivary glands of Chrysomya megacephala (Fabricius, 1794) larvae, using ultrastructural, morphological, and histological observations, and the total protein electrophoretic profile. The salivary glands present a deferent duct, originating from the buccal cavity, which bifurcates into efferent ducts that insert through a slight dilatation to a pair of tubular-shaped tissues, united in the region of fat cells. Histologically, the secretion had protein characteristics. Cell cytoplasm presented numerous free ribosomes, autophagic vacuoles, spherical and elongated mitochondria, atypical Golgi complexes, and dilated rough endoplasmic reticulum. In the apical cytoplasm, secretory granules and microvilli secretions demonstrated intense protein synthesis, basal cytoplasm with trachea insertions, and numerous mitochondria. The present work described the ultrastructure and morphology of C. megacephala third instar salivary glands, confirming intense protein synthesis and the molecular weight of soluble proteins.

Toxic effect of commercial formulations of neem oil, Azadirachta indica A. Juss., in pupae and adults of the sugarcane borer, Diatraea saccharalis F. (Lepidoptera: Crambidae) / Efeito tóxico de formulações comerciais de óleo de neem, Azadirachta indica A. Juss., em pupas e adultos da broca da cana-de-açúcar, Diatraea saccharalis F. (Lepidoptera: Crambidae)

To evaluate the toxic effect of commercial formulations of neem oil, Azadirachta indica A. Juss, pre-pupae (PP), young pupae (YP) and old pupae (OP) of Diatraea saccharalis F. (Lepidoptera: Crambidae) were sprayed with the diluted extract in distilled water at concentrations of 0.0, 0.3, 0.5, 1.0 and 2.0%. The neem extract caused concentration-dependent effects on mortality of pupae, and the pupae that failed to emerge in adults had multiple abnormalities. The longevity of pupae that emerged in adults (YP and OP group) did not differ from the control group. The abnormalities found in adults were related to mortality in all treatments, except at the concentration of 1.0%. Fertility was assessed according to the oviposition of adult females from the YP group that showed no abnormalities, through the evaluation of the number of deposited eggs and the rate of undeveloped eggs. The results showed a reduction in the number of eggs laid and an increase in the percentage of undeveloped eggs. These results show that neem oil has a high potential to control the toughest stage of the sugarcane borer and also reduces the further development. Therefore, commercial formulations of neem oil have a toxic effect on pupae and adults of D. saccharalis.(AU)

Para avaliar o efeito tóxico de formulações comerciais de óleo de neem, Azadirachta indica A. Juss, pré-pupas (PP), pupas jovens (PJ) e pupas velhas (PV) da Diatraea saccharalis F. (Lepidoptera: Crambidae) foram pulverizadas com o extrato diluído em água destilada, em concentrações de 0,0, 0,3, 0,5, 1,0 e 2,0%. O neem provocou diferentes efeitos sobre a mortalidade de pupas, dependendo da concentração. As pupas que não conseguiram emergir em adultos apresentaram anormalidades múltiplas. Quanto às pupas que emergiram em adultos (grupos PJ e PV), foi calculada a sua longevidade, que não diferiu da do grupo controle. As anormalidades encontradas em adultos estão relacionadas com a mortalidade em todos os tratamentos com exceção da concentração de 1,0%. A fecundidade foi avaliada de acordo com a oviposição de adultos fêmeas do grupo PJ, que não apresentaram anormalidades; dentro dos ovos depositados foi avaliado o número de ovos não desenvolvidos. Os resultados demonstraram redução no número de ovos depositados e aumento na porcentagem de ovos não desenvolvidos. Esses resultados mostraram que o óleo de neem tem elevado potencial para o controle do estágio mais resistente da broca da cana-de-açúcar, além de reduzir o aparecimento das fases subsequentes. Portanto, formulações comerciais de óleo de neem apresentam um efeito tóxico em pupas e adultos de D. Saccharalis.(AU)

Current progress toward vaccine and passive immunization approaches for Strongyloides spp.

Strongyloides stercoralis is a helminth parasite that can infect millions of people worldwide, particularly in tropical, subtropical and temperate regions with poor sanitation. Several aspects of epidemiology, biology and host-parasite interactions of S. stercoralis have been studied, and substantial knowledge has been acquired; however, very few studies on immunotherapeutic control strategies to prevent infection and disease in humans have been conducted. Therefore, this article reviews the current progress and targets toward vaccine and passive immunization approaches for Strongyloides spp.

Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae).

Tribolium castaneum (Coleoptera: Tenebrionidae) is a common pest of stored grains and byproducts and is normally infected by Gregarina cuneata (Apicomplexa: Eugregarinidae). The life cycle of this parasite includes the sporozoite, trophozoite, gamont, gametocyte, and oocyst stages, which occur between the epithelium and lumen of the host's midgut. This study aims to describe the morphofunctional alterations in the midgut and determine the esterase patterns in T. castaneum when parasitized by gregarines. To achieve this purpose, midguts of adult insects were isolated, processed, and analysed using light and electron microscopy. We determined total protein content, amylase activity, and the expression and related activities of the esterases by using polyacrylamide gel electrophoresis (PAGE). The midgut of T. castaneum is formed by digestive, regenerative, and endocrine cells. The effects of parasitism on the digestive cells are severe, because the gregarines remain attached to these cells to absorb all the nutrients they need throughout their development. In these cells, the most common alterations observed include expansion and fragmentation of the rough endoplasmic reticulum, development of the smooth endoplasmic reticulum, changes in mitochondrial cristae, cytoplasmic vacuolization, formation of myelin structures, spherites, large intercellular spaces, autophagic vesicles, expansion of the basal labyrinth, and cytoplasmic protrusions. Deposits of glycogen granules were also observed. Amylase activity was reduced in parasitized insects. Regenerative cells were found in disorganized crypts and did not differentiate into new cells, thus, compromising the restoration of the damaged epithelium. Though few morphological alterations were observed in the endocrine cells, results suggest that the synthesis and/or release of hormones might be impaired. Nine esterases (EST-1 to 9) were identified in the midgut of T. castaneum and were expressed in varying levels in response to parasitism. Two additional isoforms of esterases were exclusively identified in the parasitized insects. The results of this study suggest that gregarines alter the morphology and physiology of the midgut. The changes may result in nutritional depletion and the impairment of other physiological processes, such as reproduction and development of the host. Thus, further studies are needed to uncover the possibility of utilizing gregarines as biological controllers of the insect pest population.

Midgut morphophysiology in Sitophilus zeamais Motschulsky, 1855 (Coleoptera: Curculionidae).

Sitophilus zeamais is one of the most aggressive pests of stored grains, causing a significant decrease in the nutritional quality of the grains and major losses in economic trade. The foraging capacity of this pest is assigned to its highly efficient digestive system. Investigations on the morphofunctional features of the midgut, which is the most active region of the alimentary canal, are fundamental to understand the feeding habits of this species. In this study, the midgut of adult insects was isolated, processed, and analyzed on light microscopy, scanning and transmission electron microscopy, protein and enzymatic activities determination, including analyses of the starch hydrolysis products. In S. zeamais, the midgut was differentiated into anterior midgut and posterior midgut, and consisted of digestive, regenerative and endocrine cells. The anterior midgut showed high density of regenerative crypts. Cells containing organelles associated with protein synthesis and presence of amylases and lipases indicated that majority of the digestion process occurred in the anterior midgut. The posterior midgut exhibited numerous gastric caeca and peritrophic membrane. Cells with poorly differentiated cytoplasmic into organelles, elongated microvilli, and low enzymatic activities indicated that the posterior midgut was mainly involved in absorption.

Analysis of nucleus activity in malpighian tubules of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) larvae by critical electrolyte concentration.

Diatraea saccharalis (Fabricius) has cryptonephridial type Malphigian tubules (MT). This type of MT is characterized by the penetration of the distal part of the MT into the external walls of the rectum, which is usually lined with a perinephral membrane. The MT is divided into three differentiated regions: proximal, middle and distal. In this study, our objective was to compare the nuclear activities of each one of the three regions of the D. saccharalis MT by using a nuclear basophilic technique and critical electrolyte concentration with a toluidine blue stain at pH 4.0. This method allows differentiation of DNA/protein complexes in in situ and in vitro chromatin. MT chromatin structure in D. saccharalis is variable. Fifth instars have a more decondensed chromatin than fourth instars. The distal tubule region was the most decondensed region of the MT. Our data show an elevated genetic activity of the MT in the pre-metamorphosis period. The distal region of the MT has the highest observed activity, which may be associated with the re-absorption of useful components and the excretion of waste materials.

Analysis of nucleus activity in malpighian tubules of Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) larvae by critical electrolyte concentration

Diatraea saccharalis (Fabricius) has cryptonephridial type Malphigian tubules (MT). This type of MT is characterized by the penetration of the distal part of the MT into the external walls of the rectum, which is usually lined with a perinephral membrane. The MT is divided into three differentiated regions: proximal, middle and distal. In this study, our objective was to compare the nuclear activities of each one of the three regions of the D. saccharalis MT by using a nuclear basophilic technique and critical electrolyte concentration with a toluidine blue stain at pH 4.0. This method allows differentiation of DNA/protein complexes in in situ and in vitro chromatin. MT chromatin structure in D. saccharalis is variable. Fifth instars have a more decondensed chromatin than fourth instars. The distal tubule region was the most decondensed region of the MT. Our data show an elevated genetic activity of the MT in the pre-metamorphosis period. The distal region of the MT has the highest observed activity, which may be associated with the re-absorption of useful components and the excretion of waste materials.

Spherites in the midgut epithelial cells of the sugarcane borer parasitized by Cotesia flavipes.

Diatraea saccharalis, the main pest of sugarcane, has been controlled by Cotesia flavipes. Very little is known about the effect of parasitism on the host organs, including the midgut. The Lepidoptera midgut epithelium is composed of columnar, goblet, regenerative, and endocrine cells. Spherites have been described in columnar and regenerative cells of several Lepidoptera species, and presented a lot of functional meaning. We identified spherites in the midgut epithelial cells of non-parasitized D. saccharalis larvae analyzed the effect of parasitism on spherite morphology and distribution along the length of the midgut. Midgut fragments of both non-parasitized and parasitized larvae were processed for transmission electron microscopy. All the midgut epithelial cells showed spherites, but they were not preferentially located in a particular part of the cells. Parasitized larvae had more spherites, mainly in the columnar cells, than non-parasitized larvae. This observation was associated with an ionic imbalance within the insect host. Spherites were more abundant in the anterior midgut region than in other regions, which suggests that this region is involved in ion transport by intracellular and/or paracellular route. The morphological variability of spherites in the cells of parasitized larvae was related to the developmental stages of these structures.

Cytopathological process by multiple nucleopolyhedrovirus in the testis of Bombyx mori L., 1758 (Lepidoptera: Bombycidae).

A cytopathological methodology was used to analyze infection by Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), a geographic isolate of the family Baculoviridae, in the caterpillar testes of the B. mori. Japanese B. mori strain caterpillar, fifth instar, were inoculated with BmMNPV and their testes were collected and processed for light and transmission electronic microscopy. Epithelial coating cells and interfollicular septa in testes were susceptible to BmMNPV. The first evidence of infection was detected on the 6th day post-inoculation (p.i.) in the external epithelium, and on the 7th day p.i. in the internal epithelium and interfollicular septa. Cytopathological characteristics consisted of hypertrophied nuclei, the formation of virogenic stroma, and the occlusion of virions in polyhedron protein crystals in several stages of development. At the end of the infectious process, cell lysis and release of polyhedra into the extracellular medium occurred. Histopathology revealed early infection foci in the surrounding regions of tracheal insertions, thus underlining the role of the trachea as an infection-spreading organ in insects. This spreading occurs through penetration of the basal lamina, which facilitates entry of the budded virus into the testis. Additionally, an alignment of a partial sequence of the ORF 14 of the BmMNPV geographic isolate with other NPV certified the virus genera.

Esterase-3 polymorphism in the sugarcane borer Diatraea saccharalis (Lepidoptera, Pyralidae)

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and alpha- and beta-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and alpha-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3F and Est-3S, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (chi² = 2.4; p < 0.01). The negative value for the Wright's fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations