Harmful Effects of Pyraclostrobin on the Fat Body and Pericardial Cells of Foragers of Africanized Honey Bee.

Managed honey bees are daily exposed in agricultural settings or wild environments to multiple stressors. Currently, fungicide residues are increasingly present in bees' pollen and nectar and can harm colonies' production and survival. Therefore, our study aimed to evaluate the effects of the fungicide pyraclostrobin on the fat body and pericardial cells of Africanized honey bees. The foragers were divided into three experimental treatment groups and two controls: pyraclostrobin 0.125 ng/µL (FG1), 0.025 ng/µL (FG2), 0.005 ng/µL (FG3), untreated control (CTL), and acetone control (CAC). After five days of oral exposure (ad libitum), the bees were dissected and prepared for histopathological and morphometric analysis. The FG1-treated bees showed extensive cytoarchitecture changes in the fat body and pericardial cells, inducing cell death. Bees from the FG2 group showed disarranged oenocytes, peripheral vacuolization, and pyknotic nuclei of pericardial cells, but the cytoarchitecture was not compromised as observed in FG1. Additionally, immune system cells were observed through the fat body in the FG1 group. Bees exposed to FG3 demonstrated only oenocytes vacuolization. A significant decrease in the oenocyte's surface area for bees exposed to all pyraclostrobin concentrations was observed compared to the CTL and CAC groups. The bees from the FG1 and FG2 treatment groups presented a reduced surface area of pericardial cells compared to the controls and the FG3 group. This study highlighted the harmful effects of fungicide pyraclostrobin concentrations at the individual bee cellular level, potentially harming the colony level on continuous exposure.

Are native bees in Brazil at risk from the exposure to the neonicotinoid imidacloprid?

All across the world, different countries use Ecological risk assessments (ERA) of pesticides to pollinators as a regulatory tool to understand the safety of pesticide use in agriculture. However, pesticide application is still recognized as one of the main stress factors causing a decline in the global population of bees. In all ERA procedures, the effects of pesticides on the honey bee species Apis mellifera are used as a reference for the effects on all different bee species. To evaluate if tropical native bees are protected by the current risk assessment procedures and to propose improvements to the methods, we assessed the ecological risk of the neonicotinoid imidacloprid posed to native and exotic bee species. The risk was assessed through a low (TIER I) and an intermediate (TIER II) level of analysis. For TIER I the USEPA BeeREX model was used and for TIER II the Species Sensitivity Distribution (SSD) approach was adopted. For the imidacloprid exposure conditions, four different crops were taken into consideration; bean, passion fruit, sunflower and tomato. The imidacloprid risk on native species was assessed both by extrapolating the effects obtained to Apis species, and by using ecotoxicological data from tests performed with native species. In TIER I, the risks calculated through empirical data showed that more than 50% of the non-Apis species presented risk levels of 28-180% higher than those obtained with the extrapolation factor used in the Brazilian pesticide regulation. In TIER II, the SSDs showed that most of the native bees are more sensitive to imidacloprid than the Africanized A. mellifera. This is the first study in which an ERA of a pesticide was conducted on tropical bee species. Here we also present some gaps and perspectives for future studies aiming to improve the risk assessment of pesticides in terrestrial environments.

Sublethal doses of imidacloprid and pyraclostrobin impair fat body of solitary bee Tetrapedia diversipes (Klug, 1810).

Solitary bees present greater species diversity than social bees. However, they are less studied than managed bees, mainly regarding the harmful effects of pesticides present in agroecosystems commonly visited by them. This study aimed to evaluate the effect of residual doses of imidacloprid and pyraclostrobin, alone and in combination, on the fat body (a multifunctional organ) of the neotropical solitary bee Tetrapedia diversipes by means of morphological and histochemical evaluation of oenocytes and trophocytes. Males and females of newly-emerged adults were submitted to bioassays of acute topical exposure. Experimental groups were essayed: control (CTR), solvent control (ACT), imidacloprid (IMI, 0.0028 ng/µL), pyraclostrobin (PYR, 2.7 ng/µL) and imidacloprid + pyraclostrobin (I + P). The data demonstrated that the residual doses applied in T. diversipes adults are sublethal at 96 h. Both oenocytes and trophocytes cells responded to topical exposure to the pesticides, showing morphological changes. In the IMI group, the bee oenocytes showed the greatest proportion of vacuolization and altered nuclei. The pyraclostrobin exposure increased the intensity of PAS-positive labeling (glycogen) in trophocytes. This increase was also observed in the I + P group. Changes in energy reserve (glycogen) of trophocytes indicate a possible mobilization impairment of this neutral polysaccharide to the hemolymph, which can compromise the fitness of exposed individuals. Also, changes in oenocytes can compromise the detoxification function performed by the fat body. This is the first study to show sublethal effects in neotropical solitary bees and highlight the importance of studies with native bees.

Differential liver histopathological responses to amphibian chytrid infection.

Amphibians have been facing a pandemic caused by the deadly fungus Batrachochytrium dendrobatidis (Bd). Although studies have elucidated cutaneous and homeostatic disturbances, it is still unknown if the hepatic function can be affected or if hepatic effects differ among host species. Thus, we evaluated the effects of an experimental Bd infection on the liver (histopathology and the hepatosomatic index) of 2 anuran species (Xenopus laevis and Physalaemus albonotatus) with different susceptibilities to Bd infection and compared them to uninfected controls. Bd infection increased the melanomacrophage cell area and induced leukocyte infiltration in both species. The effects were more pronounced in the sensitive species, P. albonotatus, which showed severe reduction in glycogen stores and liver atrophy, due to energetic imbalance. Hepatocytes of P. albonotatus also showed ballooning degeneration (vacuolization), which could lead to cell death and liver failure. Our results provide evidence that although the sensitive species showed more severe effects, the tolerant species also had hepatic responses to the infection. These findings indicate that hepatic function can play an important role in detoxification and in immune responses to chytridiomycosis, and that it may be used as a new biomarker of health status in chytrid infections.

Neurotoxic and respiratory effects of human use drugs on a Neotropical fish species, Phalloceros harpagos.

Pharmaceutical drugs are usually and continuously carried to the aquatic environment in different ways. Thus, they are pseudo-persistent in the environment, and they may exert deleterious effects on aquatic organisms. The objective of the present study was to investigate the acute and chronic effects of two widely used pharmaceutical drugs, paracetamol (analgesic and antipyretic) and propranolol (ß-blocker) on the activity of specific biomarkers (namely cholinesterase enzymes and lactate dehydrogenase) of the neotropical fish Phalloceros harpagos. The obtained results indicate an inhibition of the activity of the enzyme lactate dehydrogenase (LDH) after acute exposure to paracetamol, and an increase in cholinesterase activity in acutely propranolol-exposed fish. Chronic exposure to both drugs did not modify the enzymatic activities. Such short-term changes in enzymatic activities may be harmful to organisms, altering the preferential pathway of energy metabolism, and may induce behavioral changes that may compromise prey capture and predator escape, and in the longer term may induce population declines.

Late effect of larval co-exposure to the insecticide clothianidin and fungicide pyraclostrobin in Africanized Apis mellifera.

Among the factors that contribute to the reduction of honeybee populations are the pesticides. These chemical compounds reach the hive through forager bees, and once there, they can be ingested by the larvae. We evaluated the effects of repeated larval exposure to neonicotinoid insecticide, both in isolation and in combination with strobilurin fungicide, at environmentally relevant doses. The total consumption of the contaminated diet was 23.63 ng fungicide/larvae (pyraclostrobin) and 0.2364 ng insecticide/larvae (clothianidin). The effects on post-embryonic development were evaluated over time. Additionally, we assessed the survival pattern of worker bees after emergence, and the pesticides' effects on the behavior of newly emerged workers and young workers. Young bees that were exposed to the fungicide and those subjected to co-exposure to both pesticides during larval phase showed behavioral changes. The insecticide, both in isolation and in combination with fungicide reduced the bees' longevity; this effect of larval exposure to pesticides was stronger in bees that were exposed only to the insecticide. Although the larvae did not have sensitivity to exposure to pesticides, they showed later effects after emergence, which may compromise the dynamics of the colony, contributing to the reduction of the populations of bees in agroecosystems.

Cholinesterases characterization of three tropical fish species, and their sensitivity towards specific contaminants.

Cholinesterases are frequent targets for toxic effects, namely by insecticides derived from phosphoric and carbamic acids. This effects allows the use of cholinesterase inhibition as a biomarker for contamination of aquatic environments by these specific chemical agents. However, cholinesterases are differently responsive to environmental contaminants, according to their different forms and locations. In addition, cholinesterases seem also to be inhibited by metals, so their use as an environmental criterion requires the prior characterization of their specific forms in each species and tissues, and the study of their sensitivity. The objective of this study was to characterize the cholinesterase isoenzymes present in the brain and dorsal muscle of three tropical fish species, namely Phalloceros harpagos (Lucinda, 2008), Pterygoplichthys pardalis (Castelnau, 1855) and Astyanax altiparanae (Garutti and Britski, 2000). In vitro assays were conducted to quantify the effect of pesticides (dimethoate and carbaryl) and metals (lead and copper) on cholinesterases activity. Although acetylcholinesterase seems to be the most prevalent and abundant form, as commonly described in vertebrates, the here-obtained results showed that three cholinesterase isoenzymes occur in tissues of the three fish species. In addition, the pesticide carbaryl caused a stronger inhibition than dimethoate. Copper caused a significantly higher cholinesterasic inhibition than lead, which is also in line with most results concerning the anticholinesterasic effects by these metals. The here obtained results allowed to conclude that acetylcholinesterase is the predominant form in all tissues from the three analyzed species. In addition, cholinesterases of these three fish were responsive to common environmental contaminants, namely metals and pesticides, similarly to what was already described for fish of temperate areas. This allows using the here proposed fish species in environmental studies for the assessment of the presence of neurotoxicants under neotropical conditions.

Behavior and histopathology as biomarkers for evaluation of the effects of paracetamol and propranolol in the neotropical fish species Phalloceros harpagos.

Pharmaceutical drugs in the aquatic environment can induce adverse effects on nontarget organisms. This study aimed to assess the short-term effects of sublethal concentrations of both paracetamol and propranolol on the fish Phalloceros harpagos, specifically light/dark preference, swimming patterns, skin pigmentation, histopathology, and liver glycogen levels. Fish were acutely exposed to sublethal concentrations of both paracetamol (0.008, 0.08, 0.8, 8, 80 mg L-1) and propranolol (0.0001, 0.001, 0.01, 0.1, 1 mg L-1) under controlled conditions. For scototaxis, a significant preference for the dark compartment was observed for the group exposed to the highest concentration of paracetamol (80 mg L-1). Propranolol exposure significantly altered the swimming pattern, especially in fish exposed to the 0.001 mg L-1 concentration. Pigmentation was reduced in propranolol-exposed fish (0.1, 1 mg L-1). The lowest concentration of propranolol (0.0001 mg L-1) induced a decrease of histochemical reaction for hepatic glycogen. These data demonstrate that pharmaceuticals can induce sublethal effects in nontarget organisms, even at low concentrations, compromising specific functions of the individual with ecological relevance, such as energy balance and behavior.

Assessment of biochemical alterations in the neotropical fish species Phalloceros harpagos after acute and chronic exposure to the drugs paracetamol and propranolol.

Over time, many pollutants of anthropogenic origin have caused the contamination of aquatic ecosystems. Among several characteristics, these compounds can reach the trophic chain, causing deleterious interactions with the biota. Pharmaceutical substances can be included in this scenario as emerging contaminants that reach the aquatic environment because of direct human and veterinary usage, and release by industrial effluents, as well as through domestic dumping of surplus drugs. The effects of these compounds on exposed organisms have been studied since the 1990s, but ecotoxicological data for such chemicals are still scarce especially concerning aquatic organisms from tropical regions. Paracetamol and propranolol were selected for this study since they are frequently found in surface waters. Paracetamol is a drug used as analgesic and antipyretic, while propranolol, a ß-blocker, is used in the treatment of hypertension. The objective of this study was to assess the toxic effects of these substances on the neotropical freshwater fish Phalloceros harpagos after acute (96 h) and chronic (28 days) exposures. In order to understand the effects of these drugs on P. harpagos, biochemical markers were selected, including the enzymes involved in oxidative stress, xenobiotic metabolism, and neurotransmission (catalase, glutathione-S-transferase, and cholinesterase activities, respectively). After acute exposure, no significant alterations were observed for catalase activity, suggesting the absence of oxidative stress. On the contrary, significant alterations in glutathione-S-transferases activity were described for the higher concentrations of both pharmaceuticals after acute exposure. In addition, acute exposure to paracetamol caused a significant increase of cholinesterase activity. None of the tested pharmaceuticals caused significant changes in catalase or cholinesterase activities after chronic exposure. Glutathione S-transferases activity was significantly increased for propranolol following chronic exposure, indicating the potential involvement of phase II detoxification pathway.

Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee.

Apis mellifera perform important pollination roles in agroecosystems. However, there is often intensive use of systemic pesticides in crops, which can be carried to the colony by forage bees through the collection of contaminated pollen and nectar. Inside the colony, pollen loads are stored by bees that add honey and several enzymes to this pollen. Nevertheless, intra-colonial chronic exposure could induce sublethal effects in young bees exposed to a wide range of pesticides present in these pollen loads. This study was aimed to both determine the survival rate and evaluate the sublethal effects on the hepato-nephrocitic system in response to continuous oral exposure to lower concentrations of neonicotinoid thiamethoxam (TXT) and picoxystrobin fungicide (PXT). Exposure to a single chemical and co-exposure to both pesticides were performed in newly emerged honeybee workers. A significant decrease in the bee survival rates was observed following exposure to TXT (0.001 ng a.i./µL) and PXT (0.018 ng a.i./µL), as well as following co-exposure to TXT+PXT/2. After five days of continuous exposure, TXT induced sub-lethal effects in the organs involved in the detoxification of xenobiotics, such as the fat body and pericardial cells, and it also induced a significant increase in the hemocyte number. Thus, the hepato-nephrocitic system (HNS) reached the greatest level of activity of pericardial cells as an attempt to eliminate this toxic compound from hemolymph. The HNS was activated at low levels by PXT without an increase in the hemocyte number; however, the mobilization of neutral glycoconjugates from the trophocytes of the fat body was prominent only in this group. TXT and PXT co-exposure induced intermediary morphological effects in trophocytes and pericardial cells, but oenocytes from the fat body presented with atypical cytoplasm granulation only in this group. These data showed that the realistic concentrations of these pesticides are harmful to newly emerged Africanized honeybees, indicating that intra-colonial chronic exposure drastically reduces the longevity of bees exposed to neonicotinoid insecticide (TXT) and the fungicide strobilurin (PXT) as in single and co-exposure. Additionally, the sublethal effects observed in the organs constituting the HNS suggest that the activation of this system, even during exposure to low concentrations of theses pesticides, is an attempt to maintain homeostasis of the bees. These data together are alarming because these pesticides can affect the performance of the entire colony.

Liver alterations in Oreochromis niloticus (Pisces) induced by insecticide imidacloprid: Histopathology and heat shock protein in situ localization.

Liver is very sensitive to environmental contaminants such as pesticides, it being the first target of toxicity of a substance. The objective of this study was to investigate the possible effects of the insecticide imidacloprid (IMI) on the liver of Oreochromis niloticus according concentrations used for growing sugarcane. A semi-quantitative analysis of histopathological alterations of IMI on liver was performed by light microscopy and cellular labeling of heat shock proteins (HSP70) by immunohistochemistry. The most common changes in liver at all concentrations of IMI were hydropic degeneration, pyknotic nuclei, and loss of cell limits. Steatosis and increased levels of HSP70 were detected in hepatocytes with the highest concentration of IMI. In conclusion, the tested concentrations of IMI induced histopathological changes in the liver of O. niloticus and active defence mechanisms to maintain the morphophysiological integrity of the liver. This insecticide has a toxicity potential for these fish, which is a non-target organism of its action.

Bilaminar Device of Poly(Lactic-co-Glycolic Acid)/Collagen Cultured With Adipose-Derived Stem Cells for Dermal Regeneration.

Several materials are commercially available as substitutes for skin. However, new strategies are needed to improve the treatment of skin wounds. In this study, we developed and characterized a new device consisting of poly(lactic-co-glycolic acid) (PLGA) and collagen associated with mesenchymal stem cells derived from human adipose tissue. To develop the bilaminar device, we initially obtained a membrane of PLGA by dissolving the copolymer in chloroform and then produced a collagen type I scaffold by freeze-drying. The materials were characterized physically by gel permeation chromatography, scanning electron microscopy, and mass loss. Biological activity was assessed by cell proliferation assay. A preliminary study in vivo was performed with a pig model in which tissue regeneration was assessed macroscopically and histologically, the commercial device Integra being used as a control. The PLGA/collagen bilaminar material was porous, hydrolytically degradable, and compatible with skin growth. The polymer complex allowed cell adhesion and proliferation, making it a potentially useful cell carrier. In addition, the transparency of the material allowed monitoring of the lesion when the dressings were changed. Xenogeneic mesenchymal cells cultured on the device (PLGA/collagen/ASC) showed a reduced granulomatous reaction to bovine collagen, down-regulation of α-SMA, enhancement in the number of neoformed blood vessels, and collagen organization as compared with normal skin; the device was superior to other materials tested (PLGA/collagen and Integra) in its ability to stimulate the formation of new cutaneous tissue.

Impact of an environmental relevant concentration of 17α-ethinylestradiol on the cardiac function of bullfrog tadpoles.

This study evaluated if a concentration of 17α-ethinylestradiol (EE2 - 10 ng L(-1) for 96 h) normally found in Brazilian surface waters exerts any impact on cardiac function of bullfrog tadpoles (25 Gosner stage), Lithobates catesbeianus. During exposure, the animals' activity level (AL -% of active individuals) was monitored twice a day. Then, the in loco heart rate (f(H) - bpm) was determined, as well as the relative ventricular mass (RVM - % of body mass). Afterwards, cardiac ventricles were mounted for isometric force recordings (CS - mN mm(-2)), and determination of the cardiac pumping capacity (CPC - mN mm(-2) min(-1)). EE2 did not affect tadpoles' AL, although it resulted in a tachycardia in animals exposed to EE2 (f(H) = 66 bpm) when compared to controls (f(H) = 52 bpm), suggesting that EE2 acts directly on the cardiac muscle of tadpoles, rather than being a result of an increased cardiac demand due to a higher activity level (i.e., avoidance response). Additionally, EE2 exerted a positive inotropic response, which resulted in a higher CPC, which occurred independently of an increase in the number of myofibrils of EE2-exposed animals, since RVM remained similar between experimental groups. Thus, the increase on cardiac demand induced by the exposure to EE2 elevates considerably the animal energy expenditure, diverting a large amount of energy that tadpoles could use for their growth and development. These alterations can make amphibians more susceptible to predators and reduce the likelihood to reach reproductive stage.

Toxicological and histopathological effects of hydramethylnon on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers.

The leaf-cut ants are important agricultural pest, because they can cause intense defoliation in plants and destroy large areas cultivated. Although there are several works for the control of these insects by examining the toxicity of natural chemical compounds on various species of ants, few are focused on analyses of morphological changes caused in the affected organs. The aim of this study was to evaluate the effects of hydramethylnon on Atta sexdens rubropilosa workers through toxicological bioassays and morphological analysis of the post-pharyngeal glands, midgut, and Malpighian tubules of these ants. Hydramethylnon dissolved either in acetone (HA) or in a mixture of acetone and soy oil (HAO) was added to the artificial diet at a concentration of 200µg/mL. The workers fed daily with the diet containing hydramethylnon showed higher mortality than the controls, especially when HAO was used. Moreover, light and electron microscopy revealed morphological alterations in the midgut and Malpighian tubules of workers treated with HA, whereas alterations of the post-pharyngeal glands were observed in the HAO-treated group. These results indicated that the presence of soy oil provided an alternate route for the ingestion of the formicide's active ingredient and corroborated previous studies that suggested a role for the post-pharyngeal glands in lipid metabolism. Our findings suggest that the oil may carry hydramethylnon to the gland lumen, resulting in lower quantity of the active ingredient in the intestinal lumen and Malpighian tubules that explains the lower degree of morphological alterations in these structures in the workers treated with HAO. These results may provide insight into the toxicological effects of hydramethylnon on leaf-cutting ants and the use of vegetable oil as an adjuvant in baits to control ants.

Effects of sublethal dose of fipronil on neuron metabolic activity of Africanized honeybees.

Fipronil is a neurotoxic insecticide that inhibits the gamma-aminobutyric acid receptor and can affect gustative perception, olfactory learning, and motor activity of the honeybee Apis mellifera. This study determined the lethal dose (LD50) and the lethal concentration (LC50) for Africanized honeybee and evaluated the toxicity of a sublethal dose of fipronil on neuron metabolic activity by way of histochemical analysis using cytochrome oxidase detection in brains from worker bees of different ages. In addition, the present study investigated the recovery mechanism by discontinuing the oral exposure to fipronil. The results showed that mushroom bodies of aged Africanized honeybees are affected by fipronil, which causes changes in metabolism by increasing the respiratory activity of mitochondria. In antennal lobes, the sublethal dose of fipronil did not cause an increase in metabolic activity. The recovery experiments showed that discontinued exposure to a diet contaminated with fipronil did not lead to recovery of neural activity. Our results show that even at very low concentrations, fipronil is harmful to honeybees and can induce several types of injuries to honeybee physiology.

Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: a morphological, immunohistochemical, biochemical, and molecular study.

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health.

Toxicological and histopathological effects of boric acid on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers.

The current study compared the toxicity of different concentrations of boric acid in adult workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae), with toxicological bioassays, and examining the dose-dependent and time-dependent histopathological changes, of the midgut, Malpighian tubules, and postpharyngeal glands. Our results revealed the importance of conducting toxicological bioassays combined with morphological analyses of the organs of ants chronically exposed to insecticides used in commercial ant baits. In vitro bioassays showed that boric acid significantly decreases the survivorship of workers regardless of concentration, whereas the morphological data suggested progressive dose-dependent and time-dependent changes in the organs examined, which were evident in the midgut. The midgut is the first organ to be affected, followed by the postpharyngeal gland and Malpighian tubules. This sequence is in agreement with the absorption pathway of this chemical compound in the midgut, its transference to the hemolymph, possibly reaching the postpharyngeal glands, and excretion by the Malpighian tubules. These progressive changes might be due to the cumulative and delayed effect of boric acid. Our findings provide important information for the understanding of the action of boric acid in ant baits in direct and indirect target organs.

Morphological alterations induced by boric acid and fipronil in the midgut of worker honeybee (Apis mellifera L.) larvae : Morphological alterations in the midgut of A. mellifera.

Morphological alterations, by means of histological and ultrastructural analysis, have been used to determine the effects of boric acid and fipronil on midgut tissues of honeybee worker, Apis mellifera L. larvae. In order to observe possible morphological alterations in the midgut, two groups of bioassays were performed. In the first one, the larvae were chronically treated with different concentrations of boric acid added to the food (1.0, 2.5 and 7.5 mg/g). In the second group, the larvae were fed with diets containing different concentrations of fipronil (0.1 and 1 microg/g) and compared with control groups without these chemical compounds. In the first bioassay, the larvae were collected on day 3 and in the second bioassay on day 4, when the mortality rate obtained in the toxicological bioassay was not very high. The larval midguts were removed and processed for morphological analyses using a light and transmission electron microscopy. We observed cytoplasmic vacuolizations, with the absence of autophagic vacuoles, and chromatinic compacting in most of the cells in the groups treated with pesticides. The morphological alterations were far greater in the larvae treated with boric acid than in the larvae treated with fipronil. Our data suggest that the midgut cell death observed was in response to boric acid and fipronil action. This study significantly improves the understanding of the toxicological effect of these insecticides from the ecotoxicological perspective.

Macromolecular array patterns of silk gland secretion in social Hymenoptera larvae.

The cocoon, produced by most holometabolous insects, is built with silk that is usually produced by the larval salivary gland. Although this silk has been widely studied in the Lepidoptera, its composition and macromolecular arrangement remains unknown in the Hymenoptera. The macromolecular array patterns of the silk in the larval salivary gland of some meliponids, wasps, and ants were analyzed with polarized-light microscopy, and they were compared with those of Bombyx mori (Lepidoptera). There is a birefringent secretion in the glandular lumen of all larvae, due to filamentous structural proteins that display anisotropy. The silk in the distal, middle and proximal regions of the secretory portion of Formicidae and Vespidae glands presented a lattice optical pattern. We found a different pattern in the middle secretory portion of the Meliponini, with a zigzag rather than a lattice pattern. This indicates that the biopolymer fibers begin their macromolecular reorganization at this glandular region, different from the Formicidae and the Vespidae, in which the zigzag optical pattern was only found at the lateral duct. Probably, the mechanism of silk production in the Hymenoptera is a characteristic inherited from a common ancestor of Vespoidea and Sphecoidea; the alterations in the pattern observed in the Meliponini could be a derived characteristic in the Hymenoptera. We found no similarity in the macromolecular reorganization patterns of the silk between the Hymenoptera species and the silkworm.

Silk formation mechanisms in the larval salivary glands of Apis mellifera (Hymenoptera: Apidae).

The mechanism of silk formation in Apis mellifera salivary glands, during the 5th instar, was studied. Larval salivary glands were dissected and prepared for light and polarized light microscopy, as well as for scanning and transmission electron microscopy. The results showed that silk formation starts at the middle of the 5th instar and finishes at the end of the same instar. This process begins in the distal secretory portion of the gland, going towards the proximal secretory portion; and from the periphery to the center of the gland lumen. The silk proteins are released from the secretory cells as a homogeneous substance that polymerizes in the lumen to form compact birefringent tactoids. Secondly, the water absorption from the lumen secretion, carried out by secretory and duct cells, promotes aggregation of the tactoids that form a spiral-shape filament with a zigzag pattern. This pattern is also the results of the silk compression in the gland lumen and represents a high concentration of macromolecularly well-oriented silk proteins.