Chemical detection of the proteins and lipids in the fat body cells from workers of Attini ants (Hymenoptera: Formicidae)

The ultrastructural cytochemistry study and the chemical analysis carried out to examine the fat body of workers of the basal Attini Cyphomyrmex rimosus and Mycetarotes parallelus and the derived Acromyrmex disciger and Atta laevigata revealed very electrondense protein granules of varied sizes and shapes distributed throughout the cytoplasm of parietal and perivisceral trophocytes and oenocytes. Lipids were present in the cytoplasm of trophocytes as very electrondense granules. In M. parallelus, A. disciger, and A. laevigata, lipids were present as droplets. In parietal as well as perivisceral oenocytes, lipid granules were very electrondense. In A. laevigata, lipids were present as droplets. The chemical analysis using gas chromatography showed that the main lipid compounds present in trophocytes and oenocytes were saturated (myristic, palmitic, and stearic) and unsaturated fatty acids (oleic and linoleic). Also, in basal species, lipid compounds consisted of mainly saturated fatty acids, while in derived species saturated as well as unsaturated fatty acids were observed.     

Roles of fat body trophocytes,mycetocytes and urocytes in the American cockroach,Periplaneta americana under starvation conditions: An ultrastructural study

In insects, trophocytes (adipocytes) are major cells of a storage organ, the fat body, from which stored glycogen and lipids are mobilized under starvation. However, cockroaches have 2 additional types of cell in the fat body: mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, and urocytes depositing uric acid in urate vacuoles. These cells have not been investigated in terms of their roles under starvation conditions. To gain insight into the roles of trophocytes, mycetocytes and urocytes in cockroaches, structural changes were first investigated in the cells associated with starvation in the American cockroach, Periplaneta americana, by light and electron microscopy. The area of lipid droplets in trophocytes, the endosymbiont population and mitotic activity in mycetocytes, and the area of urate vacuoles in urocytes were analyzed in association with survival rates of the starved cockroaches. After 2 weeks of starvation, trophocytes lost glycogen rosettes and their area of lipid droplets decreased, but almost all cockroaches survived this period. However, further starvation did not reduce the area, but the survival rates dropped rapidly and all cockroaches died in 7 weeks. Endosymbionts were not affected in terms of population size and mitotic activity, even if the cockroaches were dying. The area of urate vacuoles rapidly decreased in a week of starvation and did not recover upon further starvation. These results indicate that starved cockroaches mobilize glycogen and lipids stored in trophocytes to survive for 2 weeks and then die after the exhaustion of nutrients in these cells. Endosymbionts are not digested for the recycling of nutrients, but uric acid is reused under starvation.     

Fat body cells of Amblyomma cajennense partially engorged females (Acari: Ixodidae) and their role on vitellogenesis process

During the process of Arthropoda reproduction, the synthesis and uptake of proteins, carbohydrates, and lipids by oocytes is termed vitellogenesis. These compounds that will make up the yolk may be in ticks endogenously synthesized by the oocytes and/or exogenously produced by the fat body and pedicel cells. This study examined the fat body of Amblyomma cajennense ticks at the cytochemical ultrastructural level to demonstrate the presence of lipids, proteins and carbohydrates in trophocytes. The lipids were detected in higher quantity than proteins and carbohydrates in the fat body cells, suggesting that the role of the fat body in tick is stored lipids and carbohydrates to convert them in energy, or still they could be used with cell structural purpose. The electrophoresis technique applied at A. cajennense fat body demonstrated specifically the molecular mass of proteins: about 98 kDa. By the other hands, the fat body is not the organ responsible for the synthesis of the yolk protein, role probably performed by the pedicel cells.     

Synthesis and release of hydrocarbons by the oenocytes of the desert locust, Schistocerca gregaria

When incubated in vitro, the oenocytes in the peripheral fat body of the desert locust incorporate Na-¹⁴C-acetate into hydrocarbons (paraffins). The presence of haemolymph in the incubation medium greatly stimulates the release of the ¹⁴C-hydrocarbons into the medium. The labelled hydrocarbons appear to be rapidly released by the cells into the incubation medium as a function of time provided that haemolymph is present. The fact that the oenocytes not only synthesize ¹⁴C-hydrocarbons but also release them into the medium supports the hypothesis that the oenocytes of the desert locust synthesize cuticle lipids.     

A comparative study of fat body morphology in five mosquito species

The insect fat body plays major roles in the intermediary metabolism, in the storage and transport of haemolymph compounds and in the innate immunity. Here, the overall structure of the fat body of five species of mosquitoes (Aedes albopictus, Aedes fluviatilis, Culex quinquefasciatus, Anopheles aquasalis and Anopheles darlingi) was compared through light, scanning and transmission electron microscopy. Generally for mosquitoes, the fat body consists of lobes projecting into the haemocoel and is formed by great cell masses consisting of trophocytes and oenocytes. Trophocytes are rich in lipid droplets and protein granules. Interestingly, brown pigment granules, likely ommochromes, were found exclusively in the trophocytes located within the thorax and near the dorsal integument of Anopheles, which is suggestive of the role these cells play in detoxification via ommochrome storage. This study provides a detailed comparative analysis of the fat body in five different mosquito species and represents a significant contribution towards the understanding of the structural-functional relationships associated with this organ.     

Reproductive and worker castes in the primitively eusocial wasp Belonogaster petiolata (DeGeer) (Hymenoptera: Vespidae): evidence for pre-imaginal differentiation

Until recently, morphological differences between castes in independent-founding polistine wasps were considered absent. This paper investigates the extent of morphological and physiological differences between reproductive (foundress and gyne) and worker castes of Belonogaster petiolata, and tests the hypothesis that caste differentiation in this species occurs pre-imaginally.Foundresses were significantly larger than workers, to the extent that foundress/worker ratios were comparable with those between queens and workers in some swarm-founding Polistinae. Early emerging workers were small, but body size increased over the colony cycle such that late-season workers were similar in size to gynes. In proportion to body size, workers possessed broader heads while foundresses and gynes had broader thoraces and gasters. All queens, 98% of subordinate foundresses, and 95% of over-wintering gynes were inseminated. Workers were never inseminated and lacked mature ovaries in colonies with active queens. However, in the absence of the queen (and other foundresses), 11% of workers developed mature ovaries. Ovarian size and fat content of foundresses and gynes was significantly greater than that of workers. The differences in external morphology and reproductive physiology between castes support the hypothesis that differentiation occurs pre-imaginally. However, imaginal factors, in particular social dominance of the queen, maintain the reproductive subordinance of workers.     

Glandular Epithelium as a Possible Source of a Fertility Signal in Ectatomma tuberculatum (Hymenoptera: Formicidae) Queens

The wax layer covering the insect''s cuticle plays an important protective role, as for example, uncontrolled water loss. In social insects, wax production is well-known in some bees that use it for nest building. Curiously, mated-fertile queens of the ant Ectatomma tuberculatum produce an uncommon extra-wax coat and, consequently queens (mated-fertile females) are matte due to such extra cuticular hydrocarbon (CHC) coat that covers the cuticle and masks the brightness of the queens'' cuticle while gynes (virgin-infertile queens) are shiny. In this study, histological analysis showed differences in the epidermis between fertile (i.e., queens or gynes with highly ovarian activity) and infertile females (gynes or workers with non developed ovaries). In fertile females the epidermis is a single layer of cubic cells found in all body segments whereas in infertile females it is a thin layer of flattened cells. Ultrastructural features showed active secretory tissue from fertile females similar to the glandular epithelium of wax-producing bees (type I gland). Different hypotheses related to the functions of the glandular epithelium exclusive to the E. tuberculatum fertile queens are discussed.     

Fine Structure of the Fat Body and its Bacteroids in Blattella germanica (Blattodea)

The abdominal fat body of the cockroach Blattella germanica contains three characteristic cell types—trophocytes, bacteriocytes and urate cells—which have been investigated by electron microscopy. The trophocytes are rich in lipid droplets of different sizes; glycogen, rough endoplasmic reticulum and mitochondria are also abundant. In females immediately after eclosion, the trophocytes contain a greater number of lipid droplets, some of which have different electron density; glycogen and cytoplasmic organelles are clearly reduced. The bacteriocytes hold rod-like and spherical bacteroids, which are encapsulated by a vacuolar membrane; they show a thin cytoplasmic membrane and an evident cell wall surrounded by a membrane-like outer envelope. The bacteroids appear to be dividing either by transverse partition or by budding. The urate cells, adjacent to the bacteriocytes, are characterized by complex urate vacuoles delimited by a double layer-structure.     

Seasonal Dynamics in the Chemistry and Structure of the Fat Bodies of Bumblebee Queens

Insects’ fat bodies are responsible for nutrient storage and for a significant part of intermediary metabolism. Thus, it can be expected that the structure and content of the fat body will adaptively change, if an insect is going through different life stages. Bumblebee queens belong to such insects as they dramatically change their physiology several times over their lives in relation to their solitary overwintering, independent colony foundation stage, and during the colony life-cycle ending in the senescent stage. Here, we report on changes in the ultrastructure and lipid composition of the peripheral fat body of Bombus terrestris queens in relation to seasonal changes in the queens’ activity. Six life stages are defined and evaluated in particular: pharate, callow, before and after hibernation, egg-laying, and senescence. Transmission electron microscopy revealed that the fat body contained two main cell types–adipocytes and oenocytes. Only adipocytes reveal important changes related to the life phase, and mostly the ration between inclusion and cytoplasm volume varies among particular stages. Both electron microscopy and chemical analyses of lipids highlighted seasonal variability in the quantity of the stored lipids, which peaked prior to hibernation. Triacylglycerols appeared to be the main energy source during hibernation, while the amount of glycogen before and after hibernation remained unchanged. In addition, we observed that the representation of some fatty acids within the triacylglycerols change during the queen’s life. Last but not least, we show that fat body cell membranes do not undergo substantial changes concerning phospholipid composition in relation to overwintering. This finding supports the hypothesis that the cold-adaptation strategy of bumblebee queens is more likely to be based on polyol accumulation than on the restructuring of lipid membranes.     

Cloning of PaAtg8 and roles of autophagy in adaptation to starvation with respect to the fat body and midgut of the Americana cockroach,Periplaneta americana

Starvation, in particular amino acid deprivation, induces autophagy in trophocytes (adipocytes), the major component of the fat body cell types, in the larvae of Drosophila melanogaster. However, the fat body of cockroach has two additional cell types: urocytes depositing uric acid in urate vacuoles as a nitrogen resource and mycetocytes harboring an endosymbiont, Blattabacterium cuenoti, which can synthesize amino acids from the metabolites of the stored uric acid. These cells might complement the roles of autophagy in recycling amino acids in the fat body or other organs of cockroaches under starvation. We investigate the presence of autophagy in tissues such as the fat body and midgut of the American cockroach, Periplaneta americana, under starvation by immunoblotting with antibody against Atg8, a ubiquitin-like protein required for the formation of autophagosomes and by electron microscopy. Corresponding changes in acid phosphatase activity were also investigated as representing lysosome activity. Starvation increased the level of an autophagic marker, Atg8-II, in both the tissues, extensively stimulating the formation of autophagic compartments in trophocytes of the fat body and columnar cells of the midgut for over 2 weeks. Acid phosphatase showed no significant increase in the fat body of the starved cockroaches but was higher in the midgut of the continuously fed animals. Thus, a distinct autophagic mechanism operates in these tissues under starvation of 2 weeks and longer. The late induction of autophagy implies exhaustion of the stored uric acid in the fat body. High activity of acid phosphatase in the midgut of the fed cockroaches might represent enhanced assimilation and not an autophagy-related function.     

Histochemical and biochemical investigations concerning the function of larval oenocytes of Tenebrio molitor L. (Coleoptera,Insecta)

Summary Larval oenocytes of Tenebrio molitor were investigated histochemically. In contrast to the lipid droplets of the fat body, they did not stain with Sudan black. A positive reaction for lipoproteins appeared only after destructive oxidation with sodium hypochlorite. These lipoproteins are the remnants of degenerated membranes, as revealed by ultrastructural analysis. Polyphenols could be identified in the exocuticle of exuvia, and in the newly formed procuticle. Endocuticle, epidermis and oenocytes showed no staining reaction. In oenocytes a great amount of lipase is also present which could be detected with several Tweens as substrates. The significance of these lipases remains unclear, since only few glycerides are synthesized in the cells, as shown below. They may play a role in the extended membrane turnover observed in this cell type. In vitro incubation of oenocytes of the larval generation demonstrated that ¹⁴C-labeled acetate was only incorporated into the paraffin fraction. A negligible amount of the label was found in glycerides; wax esters were free of label. Larval epidermis is also capable of paraffin formation, but only to a small degree. Oenocytes of the imaginal generation located between the sternal epidermis cells of pupae and adults do not synthesize paraffins, but other more polar compounds not yet identified. Labeled waxes in cuticular lipids were detected only when ¹⁴C-acetate was injected into whole larvae, and the lipids extracted some hours later. Autoradiographs demonstrated that ¹⁴C-acetate was intensively incorporated into larval oenocytes, the rate varying in different cells. Incorporation into the epicuticle, probably into the wax layer, was clearly shown. Cuticulin and dense layer do not show an intensive label. The lamellated cuticle also seems to be impregnated with acetate derivatives.Supported by the Deutsche Forschungsgemeinschaft     

Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni

Protein synthesis at various times during the pregnancy cycle of G. austeni was determined by autoradiographic measurement of the incorporation of H³-leucine and H³-tyrosine into the cells of the fat body, oenocytes, milk gland and epidermis. The rate of utilization of these molecules is such that the labelled pool in the haemolymph is depleted before 0.5 hr after injection. The incorporation of both amino acids into fat body and oenocytes is high at eclosion and just after larviposition, with the incorporation of tyrosine by the oenocytes being much higher than that in the fat body. The same pattern of incorporation is observed in the epidermal cells. Label also appears in the endocuticle during the first 10 days of adult life. Except during the first 4 days following emergence, the incorporation of the two amino acids into the milk gland is very high, with periods of less intense protein synthesis at about the time of larviposition. The milk gland represents a highly efficient secretory system, with a t₅₀ of less than 30 min.     

Developmental plasticity in the queen-polymorphic ant Temnothorax longispinosus

Females of social Hymenoptera show developmental plasticity in response to varying social and environmental conditions, though some species have strong genetic influences on the form of the female reproductives. In ants, a queen polymorphism can occur in which large queens initiate new colonies on their own, while small queens enter established nests. Most queen polymorphisms studied to date originate due to genetic differences between individuals of differing form. Here, we report on the development of female form in response to social factors within the nest in the queen-polymorphic ant Temnothorax longispinosus. Three queen size morphs occur: a rare large queen with higher fat stores that can found new colonies independently, a large queen that has low fat stores and is behaviorally flexible, and a small queen that rejoins the natal nest. Both in nesting units collected from the field and those reared in the lab, queen presence during larval development led to fewer larvae developing as gynes (virgin, winged queens), and most of those gynes were the small morph. This queen effect is transferred to developing gyne larvae by close, physical interaction between queens and workers, and causes slower larval development. We conclude that gyne size, and therefore reproductive behavior, in T. longispinosus is developmentally plastic in response to queen presence. Plasticity in reproductive behavior may be an adaptive response to the nest sites utilized by this species. T. longispinosus nests predominantly in acorns and hickory nuts, which can vary dramatically from 1 year to the next. Since queens are more likely to be present in each nesting unit when fewer nest sites are available, the queen effect that results in more small gynes produced links the expression of colony-founding traits to ecological conditions across habitat patches.     

Yolk polypeptide synthesis in the fat body of Sarcophaga bullata: Localization,hormonal induction and cell-free translation

The fat body of adult Sarcophaga bullata consists of different cell-types. The yolk polypeptides (YPs) are localized in secretory granules in the cytoplasm of female trophocyte fat body cells while the oenocytes and larval fat body cells are immunonegative. An antiserum against the larval serum protein 1 of Drosophila crossreacts on immunoblotting with several polypeptide bands in the haemolymph with mol. wt ∼80 kD. This antiserum specifically reacts with some storage granules of the persisting larval fat body cells and not with the other fat body cell types. The trophocyte fat body cells of male Sarcophaga treated with 20-OH-ecdysone, display a similar granular type of immunoreaction with an anti-YP antiserum as in vitellogenic females. Moreover, 20-OH-ecdysone induced in the fat body of males, in contrast to methoprene, synthesis of mRNA coding for YPs to a level as high as that in vitellogenic females, as shown in the reticulocyte lysate cell-free system.     

Development and ultrastructure of the fat body cells and oenocytes of the Queensland fruit fly,Dacus tryoni (Frogg.)

Summary Half-way through the larval period in Dacus tryoni, the fat body cells begin to accumulate protein in the form of granules. Early in the pupal period, both the fat body cells and oenocytes become free in the body cavity. Meanwhile, an imaginal generation of hypodermal cells, while in the process of displacing the larval hypodermis, gives rise to an imaginal generation of oenocytes. Soon after, imaginal fat body cells also appear. A few days after emergence, the larval fat body cells and oenocytes disintegrate and their imaginal equivalents expand to fill the body cavity.This paper also describes the ultrastructure of the larval and imaginal fat body cells and of the imaginal oenocyte. In all three, tubular invaginations of the plasma membrane occupy the peripheral cytoplasm. At most stages, the fat body cells contain a considerable quantity of slightly distended, rough endoplasmic reticulum, which suggests that when these cells are not sequestering protein, they are secreting it into the blood. The imaginal oenocytes are packed with smooth endoplasmic reticulum, which supports other evidence that they participate in the synthesis of cuticular wax.For assistance with the electron microscopy, I thank Mr. Tony Webber and Miss Ann Miller of the Electron Microscopy Unit at Sydney University. For the loan of some sectioned material, I am grateful to Dr. D. T. Anderson.     

Vitellogenesis in the milkweed bug,Oncopeltus fasciatus Dallas (Hemiptera). A light and electron microscopic investigation

Histochemical and electron microscopic methods have revealed that there are four types of cell inclusions in the late vitellogenic oocytes of Oncopeltus. (a) Type 1 is a vacuole which seems to be contributed from the tropharium via the nutritive tubes. It is suggested that this type consists partly at least of nucleolus-like material (ribonucleoprotein) emitted from the nuclei of the Zone III trophocytes. (b) Type 2 is lipid yolk which in early stage oocytes seems to be produced in the “Balbiani body.” In the vitellogenic oocytes these lipid spheres are apparently imported by the oocyte from the haemolymph either through the follicle cells, or through the extracellular space in the follicular epithelium. (c) Type 3 is carbohydrate/protein yolk where at least part of the protein (“vitellogenic protein”) is taken up from the haemolymph, transported through the extracellular space in the follicular epithelium, and deposited into the oocyte by pinocytosis. (d) Glycogen is deposited from the early phases of vitellogenesis. The tropharium may contribute, besides Type 1 vacuoles, ribosomes, mitochondria, stacks of annulated lamellae, and “food vacuoles” to the oocytes. Specialized cells which line the tropharium and send projections toward the trophic core have been called “peripheral trophocytes.” Contrary to the regular trophocytes, they contain glycogen and an abundance of Golgi complexes.     

Host queen killing by a slave-maker ant queen: when is a host queen worth attacking?

A new colony of the slave-making ant Polyergus breviceps is initiated when a newly mated gyne invades a host nest and kills the resident queen. This process seems to result in chemical camouflage of the invading gyne and allows her to usurp the position of colony reproductive. Young, recently mated Formica gynes, however, are not attacked. To determine whether worker and/or immature presence is the basis for aggression, we placed eggs, larvae, pupae and workers from mature F. gnava queens with newly mated F. gnava queens and observed the responses of introducedP. breviceps queens. Because no newly mated gyne was attacked, we tested newly mated F. gnava queens (1) once they had produced eggs, (2) when the offspring reached the larval, pupal and callow stages of development, and (3) every 2 weeks until aggression ensued. Eventually all F. gnava queens were attacked but only 29 weeks after having mated. Thus, although offspring are the ultimate benefit from attacking an established F. gnava queen,P. breviceps queens detect mature queens using another time-dependent feature that is reliably indicative of reproductive status. The similarity of host queen hydrocarbon profiles, often correlated with reproductive status in other ant species, suggests that other compounds reflect queen fecundity and produce a kairomonal effect, or that another cue signals host queen and colony suitability. Our findings indicate P. breviceps gynes have evolved to respond aggressively to a host gyne cue that appears long after mating, preventing attacks on gynes without the workers necessary for colony founding.Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

Multiple mating and offspring quality in <Emphasis Type="Italic">Lasius</Emphasis> ants

Genetic diversity benefits for social insect colonies headed by polyandrous queens have received intense attention, whereas sexual selection remains little explored. Yet mates of the same queen may engage in sperm competition over the siring of offspring, and this could confer benefits on queens if the most successful sire in each colony (the majority sire) produces gynes (daughter queens) of higher quality. These benefits could be increased if high-quality sires make queens increase the percentage of eggs that they fertilize (unfertilized eggs develop into sons in social hymenopterans), or if daughters of better genetic quality are over-represented in the gyne versus worker class. Such effects would lead to female-biased sex ratios in colonies with high-quality majority gynes. I tested these ideas in field colonies of Lasius niger black garden ants, using body mass of gynes as a fitness trait as it is known to correlate with future fecundity. Also, I established the paternity of gynes through microsatellite DNA offspring analyses. Majority sires did not always produce heavier gynes in L. niger, but whenever they did do so colonies produced more females, numerically and in terms of the energetic investment in female versus male production. Better quality sires may be able to induce queens to fertilize more eggs or so-called caste shunting may occur wherever the daughters of better males are preferentially shunted to into the gyne caste. My study supports that integrating sexual selection and social evolutionary studies may bring a deeper understanding of mating system evolution in social insects.     

Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.