Deleterious Effects of Cymbopogon nardus (L.) Essential Oil on Life Cycle and Midgut of the Natural Predator Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

Cymbopogon nardus (citronella) essential oil (EO) has been widely used in the cosmetic and food industry due to its repellent and fumigant properties. The aim of this study was to evaluate its effects on the life cycle and midgut morphology of the natural predator Ceraeochrysa claveri. Larvae were fed on sugarcane borer eggs (Diatraea saccharalis) pretreated with citronella EO solutions (1-100 µg/mL in methanol, 5 s) or solvent and air-dried at room temperature for 30 min. Larval and pupal stage duration, the percentage of emergence of the insect, and malformed insects were recorded. One day after adults emerged from their cocoons, adult insects were used to obtain their midgut and analyzed using light microscopy. The chemical composition of C. nardus EO revealed that citronellal (25.3%), citronellol (17.9%), geraniol (11.6%), elemol (6.5%), δ-cadinone (3.6%), and germacrene D (3.4%) were the predominant compounds. Exposure to the EO produced a significant change in development duration for third instar and prepupa of the insect. The observed alterations in the lifecycle included prepupae with no cocoon formation, dead pupa inside the cocoon, and malformed adults. Several injuries in the midgut epithelium of exposed adults were registered, such as detachment of columnar cells leaving only swollen regenerative cells fixed on the basement membrane, and the formation of epithelial folds. In summary, these data suggest that C. nardus oil has adverse effects on the life cycle and midgut morphology of a beneficial predator.

Intraspecific variation in ultrastructure and secretion of the resin canals in Anacardium humile (Anacardiaceae).

The study combines a range of light and electron microscopy methods to access variation in secretion and ultrastructure in the secretory canals in the above- and belowground stems of Anacardium humile, which here serves as a model system. The aboveground stem canals show epithelial cells with ultrastructural characteristics typical of cells active in secretion, while in the belowground stems, the subcellular characteristics are typical of cells with low rates of metabolism. The secretory canals of the belowground stems show uniformity in size and shape, a large central vacuole, a cytoplasm reduced to a thin layer at the cell periphery, and a reduced population of organelles. The aboveground stem canals had voluminous nuclei with evident nucleoli, a very dense cytoplasm with free ribosomes, polyribosomes, mitochondria with developed cristae, and ellipsoid plastids with electron-opaque droplets surrounded by a periplastid reticulum. The vacuoles were of different sizes and often had membranous contents and the dictyosomes were very developed with dilated ends to the cisternae, rough endoplasmic reticulum, and numerous vesicles. The results show that particularities in above- and belowground environment have significant implications for ultrastructural morphology and functioning of secretory canals in the stems of A. humile.

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.

Midgut and fat body: Multisystemic action of pyriproxyfen on non-target organism Ceraeochrysa claveri (Neuroptera: Chrysopidae).

Morphological tools can assist in the evaluation of effects of insecticides on non-target insects. Pyriproxyfen, a juvenile hormone analog, is known to interfere with growth and metamorphosis of insects. However, there are studies showing indirect effects on natural enemies, including green lacewings. Few prior studies describe morphological effects of pyriproxyfen on target insect organs, especially on natural enemies. Through morphological tools, this study aimed to characterize the midgut and fat body, both important organs of digestion and great metabolic activity respectively, of the predator Ceraeochrysa claveri after chronic exposure to pyriproxyfen. Larvae of C. claveri were fed Diatraea saccharalis egg clusters treated with pyriproxyfen in solution of 50 or 100 mg a.i. L-1 throughout the larval stage. The biological data revealed significant increases in development time, especially in the third instar, and in cumulative mortality from the prepupal into the pupal stage. Morphological analysis of adult midgut (≤24 h old) showed damage including formation of epithelial folds, intercellular spaces, emission of cytoplasmic protrusions. Both fat body regions presented decrease of lipid droplets, vacuolization of trophocytes and mitochondrial injury featuring a multisystemic action. In both organs, pyriproxyfen exposure induced significant oxidative stress by mitochondrial superoxide production. Cytoprotective responses were induced in midgut and fat body cells by augmenting the number of cytoplasmic granules containing calcium and expression of HSP 90. Both organs proved to be efficient in presenting histopathological alterations, showing the sensitivity and applicability of this morphological tool for evaluating other insecticides in non-target organisms.

Reproductive toxicity of Novaluron in Bombyx mori (Lepidoptera: Bombycidae) and its impact on egg production.

Bombyx mori was used as a model to evaluate the reproductive toxicity of Novaluron in insects. Morphological analyses of the testes and ovaries of B. mori throughout their life cycle revealed important alterations in the germ and somatic cells involved in spermatogenesis and oogenesis. We observed in all testicular developmental phases that Novaluron affected not only the organization, distribution and development of the cysts containing male germ cells, but also the morphological features of cell death. Similar cellular characteristics were found in the treated B. mori ovaries, suggesting the occurrence of cell death in both organs, in addition to a significant reduction in oviposition of eggs by female moths. We demonstrated reproductive toxicity of Novaluron to the nontarget beneficial insect silkworm, thus providing a theoretical basis for revealing the reproductive toxicity of this insecticide to other nontarget beneficial insects.

Neem secretory cells: developmental cytology and indications of cell autotoxicity.

The neem tree (Azadirachta indica A.Juss.) contains a range of biologically active compounds-mainly triterpenoids produced in single secretory cells, which are distributed among all plant parts. Neem secretions are toxic to animal cells, triggering autolytic mechanisms that culminate in cell disruption. However, little is known about the self-toxicity of these secretions to the cells that produce them. We carried out an anatomical, histochemical, and ultrastructural investigation of neem's single secretory cells in the shoot apex and in young leaves. We evaluated the morphological changes as possible evidences of stress reactions to their own secretions. The subcellular apparatus involved in synthesis and compartmentation was consistent with hydrophilic and lipophilic secretions. Polymorphic plastids devoid of thylakoids and abundant smooth endoplasmic reticulum in the later stages of differentiation are comparable with previous reports on neem cotyledons with regard to terpenoid synthesis. However, secretions were compartmentalized within autophagic vacuoles and periplasmic spaces instead of in terpenoid vesicles. Cellular swelling, increased vesiculation, dilatation of endoplasmic reticulum cisternae, mitochondrial hypertrophy in the cristolysis process, autolytic vacuoles, and vacuolar degeneration culminating in protoplast autolysis are all consistent with early indications of autotoxicity. The signaling stress reaction mechanism was expressed as cytoplasmic deposits of calcium salt and by the expression of a 70-kDa heat-shock protein. The morphological and histochemical changes in the secreting cells are comparable with those described in animal cells exposed to neem oil. Our data provide evidence of cell damage and signaling reactions linked to these cells' own secretions before autolysis.

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.

Pest and natural enemy: how the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri react to azadirachtin exposure.

The effects of biopesticides on insects can be demonstrated by morphological and ultrastructural tools in ecotoxicological analysis. Azadirachtin-based products are widely used as biopesticides, affecting numerous insect populations. Through morphological biomarkers, this study aimed to characterize the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri after chronic exposure to azadirachtin. Larvae of S. eridania and C. claveri were fed with fresh purple lettuce leaves (Lactuca sativa) and egg clusters of Diatraea saccharalis treated with azadirachtin solution of 6 mg active ingredient (a.i.)/L and 18 mg a.i./L for 7 days, respectively. The biological data showed a significant reduction in survival and body mass in S. eridania and cytotoxic effects in the parietal and perivisceral fat bodies in both species. Ultrastructural cell damage was observed in the trophocytes of both species such as dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria. Trophocytes of S. eridania and C. claveri of the parietal and perivisceral layers responded to those injuries by different cytoprotective and detoxification means such as an increase in the amount of cytoplasmic granules containing calcium, expression of heat shock protein (HSP)70/HSP90, and development of the smooth endoplasmic reticulum. Despite all the different means of cytoprotection and detoxification, they were not sufficient to recover from all the cellular damages. Azadirachtin exhibited an excellent performance for the control of S. eridania and a moderate selectivity for the predator C. claveri, which presents better biological and cytoprotective responses to chronic exposure to azadirachtin.

Can exposure to neem oil affect the spermatogenesis of predator Ceraeochrysa claveri?

Novel biological control methods and integrated pest management strategies are basic requirements for the development of sustainable agriculture. As a result, there is a growing demand for research on the use of plant extracts and natural enemies such as the green lacewing, Ceraeochrysa claveri, as natural pest control methods. Studies have shown that although natural compounds such as neem oil (Azadirachta indica) are effective as pest control strategies, they also cause sublethal effects on nontarget insects, such as C. claveri. The aim of this study was to examine the effects of neem oil on C. claveri testes. C. claveri larvae were fed Diatraea saccharalis eggs, which were pretreated with 0.5%, 1%, and 2% neem oil. Testes were collected from larvae, pupae, and adults and analyzed using light and electron (transmission and scanning) microscopy. Changes in cellular stress and possible cell death were also determined by TUNEL assay and the marker HSP-70. The results showed that neem oil affects the organization and distribution of cysts in the testes and the normal sequence of cyst development, causing a delay in spermatogenesis in the testes of treated insects. Tests for cellular stress and DNA fragmentation indicated there was no cellular alteration in the treated groups. Although neem oil does not induce cell death or changes in HSP-70 expression, this biopesticide negatively impacts the process of spermatogenesis and could decrease the perpetuation of this species in the agroecosystem, indicating that the use of neem oil in association with green lacewings as a biological control should be carefully evaluated.

Neem oil (Azadirachta indica A. Juss) affects the ultrastructure of the midgut muscle of Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

Cytomorphological changes, by means of ultrastructural analyses, have been used to determine the effects of the biopesticide neem oil on the muscle fibers of the midgut of the predator Ceraeochrysa claveri. Insects, throughout the larval period, were fed eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% or 2%. In the adult stage, the midgut was collected from female insects at two stages of adulthood (newly emerged and at the start of oviposition) and processed for ultrastructural analyses. In the newly emerged insects obtained from neem oil treatments, muscle fibers showed a reduction of myofilaments as well as swollen mitochondria and an accumulation of membranous structures. Muscular fibers responded to those cellular injuries with the initiation of detoxification mechanisms, in which acid phosphatase activity was observed in large vesicles located at the periphery of the muscle fiber. At the start of oviposition in the neem oil treated insects, muscle fibers exhibited signs of degeneration, containing vacant areas in which contractile myofilaments were reduced or completely absent, and an accumulation of myelin structures, a dilatation of cisternae of sarcoplasmic reticulum, and mitochondrial swelling and cristolysis were observed. Enzymatic activity for acid phosphatase was present in large vesicles, indicating that mechanisms of lytic activity during the cell injury were utilized but insufficient for recovery from all the cellular damage. The results indicate that the visceral muscle layer is also the target of action of neem oil, and the cytotoxic effects observed may compromise the function of that organ.

Cytotoxic effects of neem oil in the midgut of the predator Ceraeochrysa claveri.

Studies of morphological and ultrastructural alterations in target organs have been useful for evaluating the sublethal effects of biopesticides regarded as safe for non-target organisms in ecotoxicological analyses. One of the most widely used biopesticides is neem oil, and its safety and compatibility with natural enemies have been further clarified through bioassays performed to analyze the effects of indirect exposure by the intake of poisoned prey. Thus, this study examined the cellular response of midgut epithelial cells of the adult lacewing, Ceraeochrysa claveri, to neem oil exposure via intake of neem oil-contaminated prey during the larval stage. C. claveri larvae were fed Diatraea saccharalis eggs treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval stage. The adult females obtained from these treatments were used at two ages (newly emerged and at the start of oviposition) in morphological and ultrastructural analyses. Neem oil was found to cause pronounced cytotoxic effects in the adult midgut, such as cell dilation, emission of cytoplasmic protrusions, cell lysis, loss of integrity of the cell cortex, dilation of cisternae of the rough endoplasmic reticulum, swollen mitochondria, vesiculated appearance of the Golgi complex and dilated invaginations of the basal labyrinth. Epithelial cells responded to those injuries with various cytoprotective and detoxification mechanisms, including increases in cell proliferation, the number of calcium-containing cytoplasmic granules, and HSP 70 expression, autophagic processes and the development of smooth endoplasmic reticulum, but these mechanisms were insufficient for recovery from all of the cellular damage to the midgut. This study demonstrates that neem oil exposure impairs the midgut by causing sublethal effects that may affect the physiological functions of this organ, indicating the importance of studies of different life stages of this species and similar species to evaluate the safe and compatible integrated use of biopesticides.

Effects of neem oil (Azadirachta indica A. Juss) on the replacement of the midgut epithelium in the lacewing Ceraeochrysa claveri during larval-pupal metamorphosis.

Larvae of the lacewing Ceraeochrysa claveri were fed on eggs of Diatraeasaccharalis treated with neem oil at concentrations of 0.5%, 1% and 2% throughout the larval period. Pupae obtained from treated larvae were used in the study at five days after the completion of cocoon spinning to investigate the effects of neem oil on the replacement of the midgut epithelium during the larval-pupal transition. We observed that the old larval epithelium was shed into the midgut lumen and transformed into the yellow body. Old cells from the yellow body were destroyed by apoptosis and autophagy and were not affected by neem oil. However, neem oil did affect the new pupal epithelium. Cells from treated pupae showed cellular injuries such as a loss of microvilli, cytoplasmic vacuolization, an increase of glycogen stores, deformation of the rough endoplasmic reticulum and dilation of the perinuclear space. Additionally, the neem oil treatment resulted in the release of cytoplasmic protrusions, rupture of the plasma membrane and leakage of cellular debris into the midgut lumen, characteristics of cell death by necrosis. The results indicate that neem oil ingestion affects the replacement of midgut epithelium, causing cytotoxic effects that can alter the organism's physiology due to extensive cellular injuries.

Action of neem oil (Azadirachta indica A. Juss) on cocoon spinning in Ceraeochrysa claveri (Neuroptera: Chrysopidae).

Neem oil is a biopesticide that disturbs the endocrine and neuroendocrine systems of pests and may interfere with molting, metamorphosis and cocoon spinning. The cocoon serves protective functions for the pupa during metamorphosis, and these functions are dependent on cocoon structure. To assess the changes in cocoon spinning caused by neem oil ingestion, Ceraeochrysa claveri larvae, a common polyphagous predator, were fed with neem oil throughout the larval period. When treated with neem oil, changes were observed on the outer and inner surfaces of the C. claveri cocoon, such as decreased wall thickness and impaired ability to attach to a substrate. These negative effects may reduce the effectiveness of the mechanical and protective functions of cocoons during pupation, which makes the specimen more vulnerable to natural enemies and environmental factors.

Effects of neem oil (Azadirachta indica A. Juss) on midgut cells of predatory larvae Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae).

The effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree, Azadirachta indica, on the midgut cells of predatory larvae Ceraeochrysa claveri were analyzed. C. claveri were fed on eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% and 2% during throughout the larval period. Light and electron microscopy showed severe damages in columnar cells, which had many cytoplasmic protrusions, clustering and ruptured of the microvilli, swollen cells, ruptured cells, dilatation and vesiculation of rough endoplasmic reticulum, development of smooth endoplasmic reticulum, enlargement of extracellular spaces of the basal labyrinth, intercellular spaces and necrosis. The indirect ingestion of neem oil with prey can result in severe alterations showing direct cytotoxic effects of neem oil on midgut cells of C. claveri larvae. Therefore, the safety of neem oil to non-target species as larvae of C. claveri was refuted, thus the notion that plants derived are safer to non-target species must be questioned in future ecotoxicological studies.