Genotoxic effect of two environmentally safe doses of cadmium on the hepato-nephrocytic system of Bombus atratus forager workers.

Eusocial bees are declining due to anthropogenic actions. Individuals can be exposed to contaminants like Cd, which have been found in pollen. Thus, we evaluated the potential genotoxicity of Cd to the hepato-nephrocitic system of Bombus atratus foraging workers exposed to environmentally safe doses of Cd (0.001 mg/kg and 0.003 mg/kg) during 72 h. To assess the genotoxicity of Cd, we conducted an alkaline comet assay. Doses of 0.001 mg/kg of Cd caused comets of levels 2 and 3, and 0.003 mg/kg of Cd induced comets of level 4, while controls exhibited regular nucleoids. Also, 0.003 mg/kg doses caused higher Damage Index, Damage Frequency, and Total Damage. Our results bring new evidence that 0.003 mg/kg Cd exerted genotoxicity to the HNS cells of B. atratus, probably affecting secondary metabolism and the detoxification capability of bees.

Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

The amphibian's spleen as a source of biomarkers for ecotoxicity assessment: Historical review and trends.

Amphibians are very sensitive to many environmental changes, so these animals are considered good bioindicator models for ecotoxicology. Given the importance of the amphibian spleen for hematopoietic and immune responses, this can be a key organ for the evaluation of biomarkers to monitor the health of individuals in nature or in captivity. In this systematic review, we searched databases and summarized the main findings concerning the amphibian spleen as a source of possible biomarkers applied in different scientific fields. The searches resulted in 83 articles published from 1923 to 2022, which applied the use of splenic samples to evaluate the effects of distinct stressors on amphibians. Articles were distributed in more than twenty countries, with USA, Europe, and Brazil, standing out among them. Publications focused mainly on anatomical and histomorphological characterization of the spleen, its physiology, and development. Recently, the use of splenic biomarkers in pathology and ecotoxicology began to grow but many gaps still need to be addressed in herpetological research. About 85 % of the splenic biomarkers showed responses to various stressors, which indicates that the spleen can provide numerous biomarkers to be used in many study fields. The limited amount of information on morphological description and splenic anatomy in amphibians may be a contributing factor to the underestimated use of splenic biomarkers in herpetological research around the world. We hope that this unprecedented review can instigate researchers to refine herpetological experimentation, using the spleen as a versatile and alternative source for biomarkers in ecotoxicology.

Effects of lithium and selenium in the tail muscle of American bullfrog tadpoles (Lithobates catesbeianus) during premetamorphosis.

The amphibian populations have faced a drastic decline over the past decades. This decline has been associated with the presence of contaminants in the environment, among other environmental stressors. The present study tested the responses following the exposure to lithium (2.5 mg L-1) and selenium (10µg L-1), both isolated and as a mixture, on the metabolic status of the tail muscle of premetamorphic American bullfrog (Lithobates catesbeianus) through the assessment of the total protein content, mobilization of glucose and triglycerides, and the activity of lactate dehydrogenase (LDH). The exposure followed a 21-day assay with two sampling periods (on the 7th and 21st day after the onset of exposure) to evaluate the effects over time. The group exposed to the mixture presented a statistically decreased LDH activity (P < 0.05) in both sampling periods. The presence of selenium elicited a statistically significant increase (P < 0.05) in the glucose mobilization after 7 days of exposure. After 21 days, the animals exposed to selenium presented levels of glucose mobilization comparable to the control group. The mobilization of glucose and triglycerides remained similar to the control group for the animals exposed to lithium and to the mixture in both periods of sampling (P > 0.05). The total protein content did not show any statistical difference in the treated groups throughout the experiment (P > 0.05). The presented results highlight the importance of the assessment of mixtures that can occur in the environment, since the combination of contaminants may elicit distinct toxicity compared with the effects triggered by the chemicals isolated.

Effects of mercury at field estimated concentration in brain of Bombus atratus (Hymenoptera: Bombini).

Although mercury neurotoxic effects are well known in several species, it is poorly studied in bees. Mercury contamination is increasing in several regions of the Brazilian Amazon Rainforest due to illegal and indiscriminate gold mining. Therefore, this study aimed to evaluate the effects of mercury (Hg) in brain Kenyon cells of foraging workers of Bombus atratus exposed to an average concentration (110 ppb) found in pots of honey from native bees of South America and Australia. Twenty forager workers were collected in the field (23° 34' S 47° 31' W), divided into control (n = 10) and exposed (n = 10) groups, and individually kept in special boxes for 48 h. For the exposed group, we offered Hg solution (at 110 ppb) ad libitum, while for the control group we offered water, and for both sucrose syrup at 70%. After the exposure time, the bees were crio-anesthezied at 4 °C. Brains were dissected and processed for morphological, morphometric, and histochemical analyses. Morphological results showed that the Kenyon cells of the Hg-exposed group presented both cytoplasmic vacuolization and nuclear pyknosis, which indicate cell death. These findings were corroborated by the acridine orange staining. Hg exposure also induced significant nuclear chromatin compaction in Kenyon cells. The calyces and peduncles of the mushroom bodies showed disorganization and vacuolization. In summary, these changes may imply in a severe impairment of the cognitive abilities of the bees, which could lead them to the loss of many tasks, such as foraging or even nest founding by the queen.

Metamorphic acceleration following the exposure to lithium and selenium on American bullfrog tadpoles (Lithobates catesbeianus).

To regulate the presence of contaminants in Brazilian water, the Brazilian Environmental Council (CONAMA) promulgates regulations regarding the concentrations of given compounds that are supposed to be safe for aquatic life. Considering these regulations, this study tested the effects of considered safe levels of lithium (2.5 mgL-1) and selenium (0.01 mgL-1), isolated and mixed, on the American bullfrog (Lithobates catesbeianus) tadpoles. The evaluation was done through the use of biomarkers of larval development as total wet weight (TWW), snout-vent-length (SVL), hind-limb-length (HLL), activity level (AL), histologic evaluation of the thyroid gland and the mortality rate. The tadpoles were allocated into four groups (n = 20 each): a control group (CT); a group exposed to lithium (LI), a group exposed to selenium (SE), and a group exposed to both lithium and selenium (SELI). The whole assay was carried out over 21 days, with two rounds of data collection (on 7th and 21st day) to evaluate the responses over time. A statistical reduction in the AL was observed in the tadpoles from the LI and SELI groups after 7 days of exposure, the same pattern was observed after 21 days. Histological analyses of the thyroid gland showed signs of up-regulation (i.e. statistic reduction in number and area of the follicles, as well a significant reduction in the area of the gland) in all exposed groups, which represents an endocrine response as an adaptative strategy to deal with polluted aquatic environment. The stress triggered by the polluted medium is discussed.

Metabolic, immunologic, and histopathologic responses on premetamorphic American bullfrog (Lithobates catesbeianus) following exposure to lithium and selenium.

The presence of chemicals and the destruction of freshwater habitats have been addressed as one of the reasons for the decline in the amphibians' populations worldwide. Considering the threat that these animals have been suffering in tropical regions, the present study tested if the Brazilian legislation, concerning the permissive levels of lithium and selenium in water bodies and effluents, warrants the protection of aquatic life. To do so, we assessed the metabolic, immunologic, and histopathologic alterations in liver samples of American bullfrog (Lithobates catesbeianus), at the premetamorphic stage, through biomarkers indicative of general energetic status, i.e., glucose, lipid, and protein metabolism using biochemical and histochemical approaches. The immunologic responses were assessed by the quantification of melanomacrophage centres (MMCs); the histopathologic evaluation of the liver sections was also performed. The assay was carried out over 21 days with two periods of sampling (after 7 and 21 days) to assess the effects of exposure over time. The animals were exposed to the considered safe levels of lithium (2.5 mg L-1) and selenium (10 µg L-1), both, isolated and mixed. The exposed animals showed alterations in glucose and lipid metabolism throughout the experiment. The intense presence of MMCs and histopathological responses are compatible with hepatotoxicity. The toxicity expressed by the employed animal model indicates that the Brazilian environmental legislation for the protection of aquatic life needs to be updated. With this study, we intend to provide data for better environmental policies and bring attention to sublethal effects triggered by the presence of contaminants in the aquatic environment.

Hepatotoxicity of the anionic surfactant linear alkylbenzene sulphonate (LAS) in bullfrog tadpoles.

The liver of anurans play an important role in metabolism, including detoxification, the biotransformation of molecules, and the storage of metabolites. Surfactants are part of domestic and industrial effluents. The effects of linear alkylbenzene sulfonate (LAS) on anuran liver remain unknown, however, some studies have evaluated the effects of LAS on the skin, gills, heart, testes, and liver of fishes. Here, we tested the hypothesis that LAS is hepatotoxic, promoting morphometric alterations in hepatocytes along with inflammation in the tissue, altering hepatic catabolism. We evaluated the effects of a LAS concentration that is considered environmentally safe in Brazilian inland waters on the liver of Lithobates catesbeianus tadpoles, including studies on morphology, morphometry, immunology, and metabolism. LAS exposure promoted enlargement of liver sinusoids and vacuolization of hepatocytes. Exposure to LAS also increased the area of mast cells and melanomacrophages (MMs). Additionally, LAS exposure increased hemosiderin inside MMs, suggesting alterations in the catabolism and storage of iron. Hepatocyte size increased after exposure to LAS, suggesting cytotoxic effects. Integrative analyses (i.e., morphometric, metabolic, and immunological) demonstrated hepatotoxic effects of LAS. These types of studies are key to understanding the negative effects of these substances on tadpole health, as these liver alterations impair anuran homeostasis.

Comet assay protocol for Bombus atratus fat body and pericardial cells (Hymenoptera, bombini) at a safe concentration of mercury.

The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL-1, but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL-1) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (fH - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and fH decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles.

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.

Effects of glyphosate and the glyphosate based herbicides Roundup Original(®) and Roundup Transorb(®) on respiratory morphophysiology of bullfrog tadpoles.

Glyphosate-based herbicides are widely used in agriculture and are commonly found in water bodies. Roundup Original(®) (RO) contains an isopropylamine glyphosate (GLY) salt containing the surfactant POEA, while Roundup Transorb R(®) (RTR) contains a potassium salt of GLY with unknown surfactants. Both contain different compositions of so-called "inert" ingredients, more toxic than glyphosate. Amphibian tadpoles often experience variations in O2 availability in their aquatic habitats; an ability to tolerate hypoxia can condition their survival and fitness. We evaluated the impacts of sublethal concentrations of GLY (1 mg L(-1)), RO (1 mg L(-1) GLY a.e) and RTR (1 mg L(-1) GLY a.e) on metabolic rate (V·O2 - mLO2 Kg1 h(-1)) of bullfrog tadpoles during normoxia and graded hypoxia, and related this to morphology of their skin, their major site of gas exchange. In control (CT) V·O2 remained unaltered from normoxia until 40 mmHg, indicating a critical O2 tension between 40 and 20 mmHg. GLY significantly reduced V·O2, possibly due to epidermal hypertrophy, which increased O2 diffusion distance to O2 uptake. In contrast, RTR increased V·O2 during hypoxia, indicating an influence of "inert" compounds and surfactants. V·O2 of RO did not differ from CT, suggesting that any increase in V·O2 caused by exposure was antagonized by epidermal hypertrophy. Indeed, all herbicides caused marked alterations in skin morphology, with cell and epithelium wall presenting hyperplasia or hypertrophy and chromatid rupture. In summary, GLY, RO and RTR exert different effects in bullfrog tadpoles, in particular the surfactants and inert compounds appear to influence oxygen uptake.

Hepatic effects of the clomazone herbicide in both its free form and associated with chitosan-alginate nanoparticles in bullfrog tadpoles.

The use of agrochemicals in agriculture is intense and most of them could be carried out to aquatic environment. Nevertheless, there are only few studies that assess the effects of these xenobiotics on amphibians. Clomazone is an herbicide widely used in rice fields, where amphibian species live. Thus, those species may be threatened by non-target exposure. However, nanoparticles are being developed to be used as a carrier system for the agrochemicals. Such nanoparticles release the herbicide in a modified way, and are considered to be more efficient and less harmful to the environment. The aim of this study was to comparatively evaluate the effect of clomazone in its free form and associated with nanoparticles, in the liver of bullfrog tadpoles (Lithobates catesbeianus) when submitted to acute exposure for 96 h. According to semi-quantitative analysis, there was an increase in the frequency of melanomacrophage centres, in the accumulation of eosinophils and in lipidosis in the liver of experimental groups exposed to clomazone - in its free form and associated with nanoparticles - in comparison with the control group, and the nanotoxicity of chitosan-alginate nanoparticles. The increase of melanomacrophage centres in all exposed groups was significant (P < 0.0001) in comparison to control group. Therefore, the results of this research have shown that exposure to sublethal doses of the herbicide and nanoparticles triggered hepatic responses. Moreover, these results provided important data about the effect of the clomazone herbicide and organic nanoparticles, which act as carriers of agrochemicals, on the bullfrog tadpole liver.

Hepato-Nephrocitic System: A Novel Model of Biomarkers for Analysis of the Ecology of Stress in Environmental Biomonitoring.

Bombus presents a serious global decline of populations and even loss of species. This phenomenon is complex and multifactorial: environmental degradation due to increasing cultivation and grazing areas, indiscriminate use of agrochemicals, and a plethora of xenobiotics daily discharged in the environment. We proposed that bees have an integrated cell system, which ensures protection against chemical stressors up to a certain limit. Therefore, this hypothesis was tested, exposing workers of Bombus morio to cadmium, a harmful trace metal nowadays widespread in our society. The workers were kept in BOD (26°C, RH 70%, in the dark), fed ad libitum, and divided into a control group (n = 20) and an experimental group (n = 20). For the first group, we offered 2 mL of distilled water; for the experimental groups, 2 mL of cadmium at 1 ppb. In relation to the control group, exposed bees showed that their fat body and hemocytes responded in synchronization with pericardial cells in a topographical and temporal cascade of events, where the fat body is the first barrier against xenobiotics, followed by pericardial cells. The immune cells participate throughout the process. To this system, we proposed the name of hepato-nephrocitic system (HNS), which may explain many phenomena that remain unclear in similar research with Apis mellifera and other species of bees, as shown in this paper. The bee's HNS is a system of highly responsive cells to toxicants, considered a novel parameter for the study of the ecology of stress applied in environmental management.

Cardiac adaptations of bullfrog tadpoles in response to chytrid infection.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) can result in heart failure in Bd-susceptible species. Since Bd infection generally does not cause mortality in North American bullfrogs, the aim of this work was to verify whether this species presents any cardiac adaptation that could improve the tolerance to the fungus. Thus, we analyzed tadpoles' activity level, relative ventricular mass, ventricle morphology, in loco heart frequency, and in vitro cardiac function. The results indicate that infected animals present an increase in both ventricular relative mass and in myofibrils' incidence, which accompanied the increase in myocytes' diameter. Such morphological alterations enabled an increase in the in vitro twitch force that, in vivo, would result in elevation of the cardiac stroke volume. This response requires much less energy expenditure than an elevation in heart frequency, but still enables the heart to pump a higher volume of blood per minute (i.e., an increase in cardiac output). As a consequence, the energy saved in the regulation of the cardiac function of Bd-infected tadpoles can be employed in other homeostatic adjustments to avoid the lethal effect of the fungus. Whether other species present this ability, and to what extent, remains uncertain, but such possible interspecific variability might explain different mortality rates among different species of Bd-infected amphibians.

Multiple blood meals in Anopheles darlingi (Diptera: Culicidae).

Anopheles darlingi is an important vector of human malaria in the Amazon. Adult females of this mosquito species require a blood meal to develop eggs, preferring humans to other blood sources. Although gonotrophic concordance has been described as the norm for An. darlingi, here we report An. darlingi female mosquitoes taking two or more blood meals within their first gonotrophic cycle. Only half of field-captured adult females fed one blood meal developed follicles to Christophers' stage V. This outcome is dependent on larval nutrition, as 88% of laboratory-raised well-nourished females completed the first gonotrophic cycle with only one blood meal, while less nourished females needed additional blood meals. Half of the field-captured blood-seeking An. darlingi females had follicles in intermediate (IIIa and IIIb) and final (V) stages of the gonotrophic cycle, supporting the conclusion that An. darlingi blood feed more than once during a gonotrophic cycle. Additionally, we observed females attempting to blood feed a second time during the same day. Additional studies of An. darlingi biting behavior are necessary to accurately estimate Plasmodium sp. entomologic inoculation rates throughout the An. darlingi vast geographical distribution.

Comparative ultrastructure of the mandibular gland in Scaptotrigona postica ( Hymenoptera, Apidae, Meliponini) Workers and males

Differences in the ultrastructure and function of the mandibular glands in developing workers and maturemales of the meliponine stingless bee Scaptotrigona postica suggest that there are age-dependent variationsin the contents of the secretion and glandular functions. In this work, we used transmission (TEM) andscanning (SEM) electron microscopy to examine the mandibular glands of S. postica workers of differentages and compared them with those of mature males. The gland anatomy did not vary between workers andmales. However, the ultrastructure of the gland cells changed according to the worker’s age, task, and sex.The mandibular gland cells in workers and males had a well developed smooth endoplasmic reticulum andpleomorphic mitochondria, indicating that the cells were involved in lipid synthesis. However, the secretionvaried in morphology and electrondensity between workers and males, which suggested differences in itscontents and, possibly, in glandular functions.

Morphological, chemical and developmental aspects of the Dufour gland in some eusocial bees (Hymenoptera, Apidae): a review

Morphological, chemical and developmental aspects of the Dufour gland in some eusocial bees (Hymenoptera, Apidae): a review. The present revision focused on the more recent data about the Dufour gland in some eusocial bees, taking in account general aspects of its morphology, secretion chemical nature, bio-synthetic pathway and development. Many functions have been attributed to this gland in eusocial bees, but none are convincing. With the new data about this gland, not only the secretion chemical pathway of the Dufour gland may be reasonable understood, as its function in some eusocial bees, especially Apis mellifera Linné, 1758, which has been extensively studied in the last years.

Aspectos morfológicos, químicos e do desenvolvimento da glândula de Dufour em algumas abelhas eussociais (Hymenoptera, Apidae): revisão. Esta revisão aborda os mais recentes dados sobre a glândula de Dufour em algumas abelhas eussociais, considerando aspectos gerais da sua morfologia, do seu desenvolvimento, da natureza química da sua secreção, assim como sua via bio-sintética. Muitas funções têm-se atribuído à glândula de Dufour nas abelhas eussociais, mas nenhuma suficientemente convincente. Os novos dados a respeito dessa glândula permitem não só conhecer razoavelmente bem a via bio-sintética como a função da secreção da glândula de Dufour em algumas abelhas eussociais, especialmente em Apis mellifera Linné, 1758, a qual tem sido extensivamente estudada nos últimos anos.

Programmed cell death during ovarian differentiation in queens of Apis mellifera Linné, 1758 (Hymenoptera, Apini)

In this study, we examined various aspects of ovarian development in adult honey bees queens (Apis mellifera). Caged honey bee queens showed an initial programmed germ cell differentiation that was independent of any external or environmental stimulus. In young queens, division of the stem germ cells resulted in cysts of clone cells (cystocytes) that were connected through intercellular bridges and appeared as rosettes. The cystocytes started differentiate shortly before the queens reached sexual maturity (about 5 days old). The oocyte subsequently appeared as a large, stained cell connected to parallel, double rows of smaller cells, or nurse cells. If the queens did not mate, germ cell differentiation was “switched off”, and development of the ovary was interrupted in an intermediate developmental stage, without follicular cell organization. Therefore, in such queens, there were no pevitellogenic follicles. This finding could explain why wvirgin queens rarely lay eggs and why after fecundation they require several days to start laying. The absence of previtellogenic follicles may also indicate that some stimulus is required for continuation of the vitellogenesis and ovary development.

Late Gametogenesis in Leptodactylus labyrinthicus (Amphibia, Anura, Leptodactylidae) and Some Ecological Considerations

Histological aspects of late gametogenesis in Leptodactylus labyrinthicus and of unfertilized oocytes collectes from clutches in the field were studied by light microscopy. Specimens were collected durin the reproductive period to determine why only 10 por cento of the oocytes deposited in foam nests are fertilized. Sections of ovaries and oocytes were stained with hematoxylin and eosin, mercury bromophenol blue and toluidine blue. During the reproductive phase, the ovaries were completely developed and consisted of a sack-shaped, multilobular structure, with each lobe containing many oocytes in advanced developmental stages. Atretic oocytes were also seen in the ovaries during the reproductive phase. Oocyte development in the ovaries was considered synchronous, although few oocytes were seen in the early developmental stages. There were differences in the morphology or stainning of oocytes in the ovary and of unfertilezed oocytes. Testicular development was synchronic with that of the ovary, with the testes also being fully developed during the reproductive period. Each seminiferous tubule had many cysts containing all of the phases of spermatogenesis, specially spermatids with different levels of nuclear condensation. Free spermatozoa were also observed in the lumen of the seminiferous tubule. The significant proportion of unfertilized oocytes present in many clutches may indicate that males produced an insuffucient number of spermarzoa to fertilize all of the oocytes or that females deposited additional oocytes subsequent to spawning. These unfertilized oocytes are ingested by larvae and may represent a reproductive strategy for increasing tadpole survival.