In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Anatomia Comparada dos Órgãos do Aparelho Reprodutor Masculino de 51 Espécies de AbelhasRESUMO -A anatomia dos órgãos internos do aparelho reprodutor de machos (ARM) adultos e pupas foi comparada em 51 espécies de abelhas, incluindo representantes de seis famílias. Foram obtidos quatro tipos diferentes de ARM. O tipo I está presente em machos das famílias mais basais (Colletidae, Andrenidae e Halictidae) e é caracterizado por três túbulos seminíferos por testículo, o qual é quase totalmente envolvido pela membrana escrotal. O tipo II é um tipo intermediário entre os tipos I e III e está presente em Mellitidae e Megachilidae, como também em alguns Apidae estudados, sendo caracterizado por possuir dutos deferentes pós-vesiculares fora da membrana escrotal e possuir três ou quatro túbulos seminíferos por testículo, exceto Apis mellifera L., a qual possui secundariamente um número aumentado de túbulos. O tipo III foi achado somente nos Apidae estudados e é caracterizado por apresentar os testículos e dutos genitais (exceto o duto deferente pós-vesicular) encapsulados separadamente, as glândulas acessórias são bem desenvolvidas e o duto ejaculador é calibroso, apresentando fissuras em sua parede externa, as quais podem ocorrer também no tipo II. O tipo IV está presente exclusivamente na tribo Meliponini e é caracterizado pela ausência de glândulas acessórias. PALAVRAS-CHAVE: Glândula acessória, genitália, morfologiaABSTRACT -The anatomy of the internal organs of the male reproductive apparatus (MRA) of adults and pupae was compared among 51 species of bees, including representatives of six families. Four different types of MRA were found. The type I is present in males of the less derived families (Colletidae, Andrenidae, and Halictidae) and is characterized by three seminiferous tubules per testis, which are almost completely enveloped by the scrotal membrane. The type II is an intermediary between types III and I and is present in Melittidae and Megachilidae, as well as in some Apidae studied, being characterized by post-vesicular deferent ducts outside the scrotal membrane and by three or four seminiferous tubules per testis, except for Apis mellifera L., which has a secondarily increased number of tubules. Type III was only found in the Apidae studied and is characterized by separately encapsulated testes and genital ducts (except for the post-vesicular deferent duct). Accessory glands are well developed, and the ejaculatory duct is thickened with fissures in its wall, which may also occur in the type II. Type IV is present exclusively in the tribe Meliponini, and is characterized by the absence of accessory glands.

No abstract

Rev. Brasil. Biol., 61(1) (With 1 figure) ABSTRACT Associated to the sting apparatus of the aculeate hymenopterans is found the poison gland, originated from the glands associated to the ovipositor of the non-aculeate hymenopterans and the less derived Dufour gland, homologue of the coletterial gland of other insects, and found in all hymenopteran females. The Dufour gland functions is mostly uncertain in hymenopterans but in ants it is involved with communication and defense and in non social bees with the nest building and protection. In wasps possibly with kin-recognition. Differences in morphology and chemical composition of the gland secretion were observed among species, in the same species, between the castes in the social species and among individual of the same caste playing different tasks or belonging to different nest. Its original function of egg-protective substance producing, or favoring the oviposition, appear to have been replaced or complemented in hymenopterans by the production of semiochemicals with function in communication.Key words: Dufour gland, hymenopterans, morphology, pheromones, ultrastructure. RESUMO Glândula de Dufour nos himenópteros (Apidae, Formicidae, Vespidae): uma revisãoAnexas ao aparelho do ferrão dos himenópteros aculeados encontram-se as glândulas de veneno e as de Dufour. A glândula de veneno é originada das glândulas associadas ao ovopositor dos himenópteros ancestrais não aculeados, já a glândula de Dufour é menos derivada, homóloga das glândulas colateriais dos outros insetos, sendo encontrada em todas as fêmeas dos himenópteros. Nestes insetos sua função é, em grande parte, desconhecida, mas, em formigas, parece estar envolvida com a comunicação e a defesa e, nas abelhas não sociais, com a construção e a proteção do ninho. Nas vespas pode estar relacionada ao reconhecimento parental. Foram observadas diferenças morfológicas e na composição química da secreção da glândula de Dufour entre as espécies, bem como na mesma espécie, entre as castas dos himenópteros sociais e entre indivíduos da mesma casta desempenhando diferentes funções ou pertencentes a ninhos diferentes. Portanto, nos himenópteros, sua função original de produzir substâncias para proteger os ovos ou favorecer a ovoposição parece ter sido substituída ou complementada com a função de produzir semioquímicos com função na comunicação.Palavras-chave: feromônios, glândula de Dufour, himenópteros, morfologia, ultra-estrutura.

The evolutionary origin of beetle bioluminescence is enigmatic. Previously, weak luciferase activity was found in the non-bioluminescent larvae of Tenebrio molitor (Coleoptera: Tenebrionidae), but the detailed tissular origin and identity of the luciferase-like enzyme remained unknown. Using a closely related giant mealworm, Zophobas morio, here we show that the luciferase-like enzyme is located in the Malpighi tubules. cDNA cloning of this luciferase like enzyme, showed that it is a short AMP-ligase with weak luciferase activity which diverged long ago from beetle luciferases. The results indicate that the potential for bioluminescence in AMP-ligases is very ancient and provide a first reasonable protoluciferase model to investigate the origin and evolution of beetle luciferases.

Utilizando cromatografia gasosa acoplada à espectrometria de massa, descrevemos as diferenças na composição química da secreção da glândula de Dufour de rainhas virgens e fisogástricas de Melipona bicolor. A secreção da glândula de rainhas virgens consiste de hidrocarbonetos, enquanto a das rainhas fisogástricas contém, além de hidrocarbonetos, uma variedade de outros compostos, tais como ésteres isobutíricos e acéticos. Tais diferenças devem indicar o estado de fecundidade da rainha e os compostos oxigenados da secreção das fisogástricas podem ajudar aumentar sua atratividade.We describe differences in the chemical composition of the Dufour gland secretion of virgin and physogastric queens of Melipona bicolor through gas chromatography and mass spectrometry. The Dufour gland secretion of virgin queens consists only of hydrocarbons, while that of physogastric queens contains, besides these, a variety of other compounds, such as isobutyrate and acetate esters. Such differences may indicate the queen fecundity condition and the oxygenated compounds of the physogastrics secretion may help to increase their attractiveness.Keywords: Dufour gland, Melipona bicolor, physogastric queen, stingless bee, pheromone, ester, hydrocarbon IntroductionThe Dufour gland is found in all female hymenopterans. In bees, the gland is located at the base of the sting apparatus, ventrally to the poison gland, and opens into the dorsal vaginal wall.1 A range of substances, from macrolactones and terpenoid esters to hydrocarbons and triglycerides, has been found in the Dufour gland secretion of bees. 2-13In solitary bees, which build underground nests, the Dufour gland produces hydrophobic substances used to line the nest 9 and to protect eggs against extreme fluctuations in humidity and against microorganisms. 14 This lining also maintains a stable environment during the several phases of development of the immature bee. 14 In the eusocial Apidae, the function of this gland is unknown. In Bombus, the Dufour gland secretion consists of hydrocarbons and esters, and is colony-and speciesspecific. 15,16 In Apis mellifera, the secretion is composed of hydrocarbons in workers, and in egg-laying workers and queens, hydrocarbons and esters. 17 Bioassays done first by Abdalla and Cruz-Landim 18 demonstrated that the virgin queen secretion provokes worker attraction. On the contrary, the worker secretion repulses their nestmate workers. Secretion of non-nestmate bees has no effect on the worker behavior. Recently, noticed that the esters of the Dufour gland secretion is the active fraction, being the egg-laying worker and queen secretion greatly attractive to nestmate workers. The non egg-laying worker gland secretion has no effect on this aspect of worker behavior.In stingless bees (Meliponini), in only one species, Nanatrigona testaceicornis workers, the chemical composition of the Dufour gland secretion has been studied. In this species 64% of the secretion consisted of the ester diterpene all-trans-geranylgeranyl acetate. 20The Dufour gland is...

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.

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 fieldcaptured 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. Journal of Vector Ecology 37 (2): 351-358. 2012.