Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.     

Preparation of Avocado Mitochondria Using Self-Generated Percoll Density Gradients and Changes in Buoyant Density during Ripening

Mitochondria from avocado (Persea americana Mill, var. Fuerte and Hass) can be rapidly prepared at every stage of ripening using differential centrifugation and self-generated Percoll gradients. The procedure results in improved oxidative and phosphorylative properties, especially for mitochondria isolated from preclimacteric fruits.

A gradual change in the buoyant density of avocado mitochondria takes place during ripening. Climacteric and postclimacteric avocado mitochondria have the same buoyant density as other plant mitochondria (potato, cauliflower), whereas mitochondria from preclimacteric fruit have a lower density. The transition in buoyant density occurs during the climacteric rise, and two populations of intact mitochondria (p = 1.060 and p = 1.075) can be separated at this stage. Evidence indicates that the difference in mitochondrial buoyant density between preclimacteric and postclimacteric mitochondria is likely due to interactions with soluble cytosolic components.

     

Ontogeny of body density and the swimbladder in yellowtail kingfish Seriola lalandi larvae

The ontogeny of larval body density and the morphological and histological events during swimbladder development were investigated in two cohorts of yellowtail kingfish Seriola lalandi larvae to understand the relationship between larval morphology and body density. Larvae <3 days post hatch (dph) were positively buoyant with a mean ± s.d . body density of 1·023 ± 0·001 g cm^?3. Histological evidence demonstrated that S. lalandi larvae are initially transient physostomes with the primordial swimbladder derived from the evagination of the gut ventral to the notochord and seen at 2 dph. A pneumatic duct connected the swimbladder to the oesophagus, but degenerated after 5 dph. Initial swimbladder (SB) inflation occurred on 3 dph, and the inflation window was 3–5 dph when the pneumatic duct was still connected to the gut. The swimbladder volume increased with larval age and the epithelial lining on the swimbladder became flattened squamous cells after initial inflation. Seriola lalandi developed into a physoclist with the formation of the rete mirabile and the gas‐secreting gland comprised low‐columnar epithelial cells. Larvae with successfully inflated swimbladders remained positively buoyant, whereas larvae without SB inflation became negatively buoyant and their body density gradually reached 1·030 ± 0·001 g cm^?3 by 10 dph. Diel density changes were observed after 5 dph, owing to day time deflation and night‐time inflation of the swimbladder. These results show that SB inflation has a direct effect on body density in larval S. lalandi and environmental factors should be further investigated to enhance the rate of SB inflation to prevent the sinking death syndrome in the early life stage of the fish larvae.     

Mitochondrial structure and dynamics as critical factors in honey bee (Apis mellifera L.) caste development

The relationship between nutrition and phenotype is an especially challenging question in cases of facultative polyphenism, like the castes of social insects. In the honey bee, Apis mellifera, unexpected modifications in conserved signaling pathways revealed the hypoxia response as a possible mechanism underlying the regulation of body size and organ growth. Hence, the current study was designed to investigate possible causes of why the three hypoxia core genes are overexpressed in worker larvae. Parting from the hypothesis that this has an endogenous cause and is not due to differences in external oxygen levels we investigated mitochondrial numbers and distribution, as well as mitochondrial oxygen consumption rates in fat body cells of queen and worker larvae during the caste fate-critical larval stages. By immunofluorescence and electron microscopy we found higher densities of mitochondria in queen larval fat body, a finding further confirmed by a citrate synthase assay quantifying mitochondrial functional units. Oxygen consumption measurements by high-resolution respirometry revealed that queen larvae have higher maximum capacities of ATP production at lower physiological demand. Finally, the expression analysis of mitogenesis-related factors showed that the honey bee TFB1 and TFB2 homologs, and a nutritional regulator, ERR, are overexpressed in queen larvae. These results are strong evidence that the differential nutrition of queen and worker larvae by nurse bees affects mitochondrial dynamics and functionality in the fat body of these larvae, hence explaining their differential hypoxia response.     

菜蛾盘绒茧蜂主要寄生因子导致的寄主小菜蛾幼虫脂肪体结构的变化

在不同的寄生状态下,菜蛾盘绒茧蜂Cotesia plutellae不同的寄生因子可引起寄主小菜蛾Plutella xylostella幼虫脂肪体结构发生相应的改变。显微和亚显微形态结构显示： 假寄生后多分DNA病毒和毒液对脂肪体结构的完整性没有显著影响,但细胞内脂质体变得小而密集,线粒体和内质网丰富,并有糖原积累; 正常寄生后,脂肪体结构被破坏,多数线粒体内嵴紊乱,脂质体也变得不规则,特别是当幼蜂完成在寄主体内发育时,寄主体内几乎无完整脂肪体存在。与此同时,同批未被寄生的小菜蛾幼虫发育到4龄末期时,体内脂肪体细胞发育正常,已开始向蛹期细胞形态转化,细胞内脂质体很大,细胞器数量较多、糖原积累丰富, 而且部分细胞已成为游离态细胞。由此证明,寄生蜂携带的寄生因子,如多分DNA病毒、毒液、畸形细胞和幼蜂等,均对寄主脂肪体结构的改变产生影响,但程度明显不同。     

NONSULFATED SULFAKININ CHANGES METABOLIC PARAMETERS OF INSECT FAT BODY MITOCHONDRIA

We investigated the effect of neuropeptide, the nonsulfated sulfakinin (SK) Zopat‐SK‐1 (pETSDDYGHLRFa) on the mitochondrial oxidative metabolism in the Zophobas atratus larval fat body. Mitochondria were isolated from beetle fat bodies 2 and 24 h after hormone injection. The administration of 20 pmol of Zopat‐SK‐1 to feeding larvae led to decreased mitochondrial oxidative activities in larval fat body. Diminished activities of citrate synthase and the cytochrome pathway, that is, nonphosphorylating and phosphorylating respiration during succinate oxidation, were observed. However, the effect of Zopat‐SK‐1 was more pronounced in fat body of insects after 24 h since hormone application. In hormone‐treated larval fat bodies, mitochondrial respiration was decreased at the level of respiratory chain and the TCA cycle as well as at the level of mitochondrial biogenesis, as indicated by decreased activities of mitochondrial marker enzymes in fat body homogenates. The inhibition of succinate oxidation may indicate the role of Zopat‐SK‐1 in the regulation of mitochondrial complex II activity. Moreover, decreased respiratory chain activity was accompanied by the reduced activity of mitochondrial energy‐dissipating pathway, uncoupling protein 4. The observed decrease in mitochondrial oxidative metabolism may reflect the Zopat‐SK‐1‐induced reduction in the metabolic rate of larval fat body linked to actual energetic demands of animal.     

GENESIS OF MITOCHONDRIA IN INSECT FAT BODY

Electron microscopy and stereological methods have been used to study the time course and mechanism of mitochondrial genesis in the adult fat body of Calpodes ethlius, (Lepidoptera, Hesperiidae). Most of the larval mitochondria are destroyed during a phase of autolysis shortly before pupation, so that pupal and early adult fat body cells have few mitochondria. The number of mitochondria per cell increases rapidly at the end of the 1st day after the adult emerges. Characteristic partitioned mitochondria appear during the period when the number is rapidly increasing. This evidence, coupled with the results of morphometric analyses of mitochondrial diameter, volume, and surface area, confirms the view that the genesis of adult mitochondria involves the growth and division of mitochondria surviving from the larva.     

Drosophila Clueless Is Highly Expressed in Larval Neuroblasts,Affects Mitochondrial Localization and Suppresses Mitochondrial Oxidative Damage

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu’s specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu’s role in mitochondrial function is not critical during larval development, but is important for pupae and adults.     

Geographic variation in competitive ability in Drosophila melanogaster

The aim of this study was to examine the latitudinal variation in preadult competitive ability of Drosophila melanogaster. Two pairs of populations from Queensland and Tasmania, Australia, were examined. Queensland flies are genetically smaller and develop more slowly than the Tasmanian flies. Survival and body size of flies raised at different temperatures and densities were compared when larvae were challenged with a common competitor. No latitudinal variation in larval survival was detected. Body size (measured as wing length) decreased with increasing temperature and larval density. Flies from the Tasmanian populations were more sensitive to the effects of temperature and density and to the joint effect of increased temperature and density. This could explain the evolution of greater growth efficiency and larger body size at lower temperatures.     

Allometric growth and functional development of the gut in developing cod Gadus morhua L. larvae

The development of the gut epithelium in cod Gadus morhua was studied during the larval period in intensive rearing systems. Cod larvae were fed enriched rotifers from mouth opening. On 17 days post‐hatch (dph) one group of larvae were fed Artemia sp. nauplii while another group were fed both rotifers and a formulated diet (co‐fed). At the end of the experiment (30 dph) larvae receiving live feed were almost three times larger than the co‐fed larvae, although no clear signs of pathological effects due to feeding regime were found in any larvae sampled for morphological studies. The midgut volume in larvae fed live feed increased by a factor of 38 during the experiment, and in particular volume increased rapidly between 24 and 30 dph. The enterocyte size increased between 12 and 24 dph from 652 ± 64 to 1479 ± 144 μm³ (mean ±s .e .). When enterocytes reached their maximum size, several morphological changes in the gut epithelium were initiated, such as increased number of mitochondria per enterocyte, increased size of the nuclei and a considerable increase in microvilli surface area. The mitochondrial membrane structures changed during the experiment, suggesting a maturation process of the mitochondria. The midgut development was strongly related to larval size rather than age. On 30 dph co‐fed larvae were equal in size to Artemia sp. fed larvae on 24 dph. This was reflected by equal values of estimated midgut volume, midgut length and total number of enterocytes and the number of mitochondria per enterocyte. The microvilli surface area, however, was significantly larger in co‐fed larvae on 24 dph compared to live‐feed larvae on 30 dph. This increase in absorptive surface was probably a response to suboptimal feeding conditions. The strong correlation between gut development and larval size and the lack of clear pathological effects, suggested that the gut tissue is flexible and can withstand periods of suboptimal nutrition at this stage.     

Differential expression of antioxidant system genes in honey bee (Apis mellifera L.) caste development mitigates ROS-mediated oxidative damage in queen larvae

The expression of morphological differences between the castes of social bees is triggered by dietary regimes that differentially activate nutrient-sensing pathways and the endocrine system, resulting in differential gene expression during larval development. In the honey bee, Apis mellifera, mitochondrial activity in the larval fat body has been postulated as a link that integrates nutrient-sensing via hypoxia signaling. To understand regulatory mechanisms in this link, we measured reactive oxygen species (ROS) levels, oxidative damage to proteins, the cellular redox environment, and the expression of genes encoding antioxidant factors in the fat body of queen and worker larvae. Despite higher mean H₂O₂ levels in queens, there were no differences in ROS-mediated protein carboxylation levels between the two castes. This can be explained by their higher expression of antioxidant genes (MnSOD, CuZnSOD, catalase, and Gst1) and the lower ratio between reduced and oxidized glutathione (GSH/GSSG). In worker larvae, the GSG/GSSH ratio is elevated and antioxidant gene expression is delayed. Hence, the higher ROS production resulting from the higher respiratory metabolism in queen larvae is effectively counterbalanced by the up-regulation of antioxidant genes, avoiding oxidative damage. In contrast, the delay in antioxidant gene expression in worker larvae may explain their endogenous hypoxia response.     

Regulation of damseifly populations: the effects of larval density on larval urvival, development rate and size in the field

SUMMARY. 1. The popuhttion density of Coenagrion pttella larvae was monitored in five populations, and of Ischntira elegans in two populations, between October 1982 and May 1983.
2. There was no measurable mortality of larvae over winter and no larval growth until April. Larvae in high density populations were smaller than those in low density populations and were more likely to have a semi- voltine life history.
3. The population density of C. ptiella was also monitored (more frequently) in two populations with differenl initial densities between July and November 1983. In the high density population there was a constant rate of larval mortality, while in the low density population there was no detectable larval mortality, indicating that larval mortality may be density dependent. Larvae in the high density population were again smaller, and more likely to be semi-voitine, than those in the low density population.
4. The role of density dependent larval growth, development and mortality in the regulation of damseifly populations is discussed.     

Intraspecific competition of Glyptotendipes paripes (Diptera: Chironomidae) larvae under laboratory conditions

Glyptotendipes paripes larvae were reared in wells of tissue culture plates, in groups of 2, 4, 8, 16, and 32 (representing densities of about 1,300, 2,600, 5,200, 10,400, and 20,800 larvae per m², respectively). Larval groups were supplied with one of two concentrations (low or high) of food and larvae were individually observed to evaluate the effects of density on mortality, growth, development, behavior, and adult body size. Increased larval densities resulted in higher mortality, as well as slower larval growth and development. The distribution of developmental time became flatter at higher density, with a wider range of values, or even became bimodal. This was a consequence of the most rapidly developing individuals at higher densities emerging as adults sooner than the fastest developing individuals at lower densities, although overall mean developmental time was longer at higher densities. At higher densities, growth and development of smaller larvae were slowed, based on the relative difference in body length between competitors. When larger competitors emerged as adults or died, the growth of smaller larvae may have accelerated, resulting in increased variability of developmental times. The effect of larval density on adult body size was complex, with the largest body size found at the lowest density and a second peak of adult size at high-middle densities, with smaller adult body sizes found at low-middle, and high densities. Similarly, as with developmental time, the range of body size increased with increasing density. Examined food concentrations had no effect on larval mortality, but significantly affected developmental time, growth rate, and adult body size. At higher densities, larvae spent more time gathering food and were engaged in aggressive or antagonistic behaviors.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Differentiated egg size of the cannibalistic salamander <Emphasis Type="Italic">Hynobius retardatus</Emphasis>

Larvae of the salamander, Hynobius retardatus, are carnivorous, and even though there are two morphs, a typical morph and a broad-headed or “cannibal” morph, both are cannibalistic. They also sometimes eat other large prey, for example larvae of the frog, Rana pirica. In natural habitats, use of both conspecific and R. pirica larvae as food may contribute more strongly to high survival and substantially to fitness when larval densities are higher, because early-stage H. retardatus larvae sometimes experience scarcity of their typical prey. In cannibalistic oviparous amphibians, larger individuals that developed from larger eggs can more efficiently catch and consume larger prey and thus their survival may be better than that of smaller individuals developed from smaller eggs. Populations might therefore diverge in respect of egg size in response to variation in the density of conspecific and R. pirica larvae in natural ponds, with eggs being larger when larval density is higher. I examined how variance in hatchling size correlated with the incidence of cannibalism, and whether increasing larval density in natural ponds correlated with increasing egg size. Variance in initial larval body size facilitated cannibalism, and egg size increased as larval density in the ponds increased. In ponds with high larval density, where cannibalism and large prey consumption is a critical factor in offspring fitness, the production of fewer clutches with larger eggs, and thus of fewer and larger offspring, results in greater maternal fitness. Variation among the mean egg size in populations is likely to represent a shift in optimum egg size across larval density gradients.     

Intranuclear disease of uncertain etiology in larvae of the blackfly,Simulium vittatum

An intranuclear disease of uncertain etiology has been found in larvae of the blackfly, Simulium vittatum (Diptera: Simuliidae). The disease, which causes little mortality, is characterized by a lack of pigmentation in the cuticle and the occurrence of melanotic deposits in the cytoplasm of fat body and midgut epithelial cells. A single, dense lacunate inclusion develops within the nuclei of these cells, ranging from 20 to 30 μm in midgut epithelial cells, and averaging 18 μm in fat body cells. The disease has been reproduced in the laboratory by feeding first-instar blackfly larvae on homogenates of diseased larvae. However, attempts to isolate a causative agent have not been successful. The cytopathology of the disease resembles the cytopathology caused by densonucleosis viruses, but particles, either free or in aggregations, typical of the virions of this virus type have not been identified in infected nuclei.     

Pleistophora oncoperae sp.n. (Protozoa: Microsporida) from Oncopera alboguttata (Lepidoptera: Hepialidae) in Australia

Pleistophora oncoperae sp.n. is described from adults and larvae of Oncopera alboguttata and O. rufobrunnea. The main site of infection was muscle, though fat body and connective tissue were also infected. Fresh pansporoblasts measured about 25 μm in diameter and contained 16 to 32 or more spores with a mean size of 5.9 × 3.1 μm. Macrospores measuring 7.7 × 4.4 μm were also seen. The mean polar filament length was 158 μm; ultrastructural studies showed that the filament is normally arranged in 14 coils (range, 13 to 20) at an angle of 53.5° to the axis of the spore. The species was found to be distinct from all previously described Pleistophora reported from Lepidoptera.     

Arginine transport into fat body mitochondria of the blowfly,Aldrichina grahami,during larval growth

Osmotic swelling tests show that neither arginine⁺ nor K⁺ passes into the matrix of nonrespiring fat body mitochondria isolated from 4 day old larvae and that arginine⁺ or K⁺ passes through the membrane of nonrespiring fat body mitochondria of 6 day old larvae. This means that the selective transport system of small ions in the fat body mitochondrial membrane is lost after the larva stops eating. Thus, the arginine transport system in the fat body mitochondria of 4 day old larvae was examined. When succinate was added to arginine solutions in which mitochondria were suspended, arginine⁺ readily entered the mitochondria if a permeant proton-yielding anion such as phosphate or acetate was present. However, permeant anions such as nitrate or chloride, which pass through the membrane without carrying protons, do not support arginine⁺ entry. Antimycin, FCCP and DNP completely inhibited the succinate-induced swelling in the solutions containing arginine phosphate and acetate. The succinate-induced swelling in the solutions containing arginine phosphate and acetate was not affected by NEM and oligomycin. The driving force for entry of arginine⁺ is concluded to be a negative inside-transmembrane potential produced when proton-conducting anions enter mitochondria to relieve the alkaline-inside pH gradient generated by electron transport. It is postulated that arginine⁺ is transported by a specific electrogenic uniport carrier.     

Effect of high sucrose concentrations on mitochondria: Analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde

It has been shown previously that intact rat liver mitochondria can be separated into two populations (designated B2 and B3) with mean buoyant densities of 1·184 and 1·216 respectively, by isopycnic sucrose density gradient centrifugation. A comparison has been made of some properties of these mitochondrial fractions from density gradients with non-fractionated mitochondria. Use was made of density gradient centrifugation for analysis of preparations fixed with appropriate concentrations of glutaraldehyde. The permeability of the membranes of non-fractionated mitochondria to sucrose was increased by exposure to hypoosmotic sucrose solutions. The B3 mitochondria differed from the non-fractionated mitochondria in their response to changes in osmotic pressure of the suspending medium while the B2 mitochondria showed essentially identical behaviour with the controls. However, under conditions of energized swelling the B2 mitochondria were markedly different to the controls. This difference, which is attributed to reduced permeability of the mitochondrial membranes to metabolites brought about by exposure to the high concentrations of sucrose encountered in the density gradient, was reversed by incubation in hypo-osmotic sucrose solutions in the presence of oxidizable substrate and permeant ions.Died December, 1969.     

Density-dependent population dynamics in larvae of the dragonfly Pachydiplax longipennis: a field experiment

Summary Several features of dragonfly population biology suggest that population regulation occurs in the larval stage. This study was designed to determine if density-dependent interactions among larval odonates can affect survival, growth and emergence. First-instar larvae of the dragonfly Pachydiplax longipennis were raised in outdoor experimental ponds at initial densities of 38, 152, and 608 larvae · m^-2, under two levels of food availability. Food availability was supplemented in half the pools by volumetric addition of zooplankton every other day. Pools in the low food treatment did not receive the zooplankton supplement.There was a strong negative effect of density on the mean growth rate of survivors, which included both emerging tenerals and individuals overwintering in the larval stage. A higher proportion emerged from low density than high density pools. Metamorphs from high density populations were smaller and emerged slightly later than those from low density, but the absolute number of metamorphs did not differ significantly among density treatments. Food supplementation significantly increased the proportion of overwintering larvae. There were no significant food-by-density interactions, indicating that food and density acted independently on larval population dynamics. Density-dependent mechanisms can clearly contribute to odonate population regulation, especially by controlling the number of larvae which emerge and the average age at reproduction. Population-level responses to density may be a result of interference among larvae.