Aeshna Cyanea Research Articles

Differential absorption and esterification of dietary long‐chain fatty acids by larvae of the dragonfly, Aeshna cyanea

In order to evaluate whether dietary long-chain fatty acids were differentially absorbed, Aeshna cyanea larvae received 5 μl oral doses containing combinations of two radiolabeled fatty acids at nearly equal radioactive and nmolar concentrations: (1) 3H-oleic and 14C-palmitic acids; (2) 3H-oleic and 14C-stearic acids; and (3) 3H-palmitic and 14C-stearic acids. After 3 h or 1 day, hemolymph samples, midgut tissue, midgut contents and fat body tissue were collected and assayed for labeled fatty acids. The 3H/14C ratios indicated that there was a preference for absorption of the monounsaturated oleic acid over both saturated palmitic and stearic acids and that the shorter palmitic acid was absorbed at a higher rate than the longer stearic acid. There were also differences in the 3H/14C ratios of the various lipid classes of the midgut wall, hemolymph, and fat body that reflected differential esterifications and transport of these fatty acids. Arch. Insect Biochem. Physiol. 40:183–193, 1999. © 1999 Wiley-Liss, Inc.

Early Morning Oviposition of Dragonflies with Low Temperatures for Male-Avoidance (Odonata: Aeshnidae, Libellulidae)

The relationship between oviposition by females [FF] and the beginning of male [M] flight activity, on the one hand, and air temperature [T] and time of day, on the other, was investigated in Northern Germany, in Southern France and in Japan. FF of some Anisoptera species, as FF of Orthetrum cancellatum (Linnaeus 1758) [Libellulidae], Aeshna cyanea (Muller 1764) [Aeshnidae], and A mixta Latreille 1805 [Aeshnidae], avoid further matings by appearing before the MM at oviposition sites, when air TT are still low. In contrast, the FF of Anax imperator Leach 1815 [Aeshnidae] are able to defend themselves against MM successfully. FF of the Aeshnidae in S-France and in Japan fly to oviposition with higher air temperatures than the FF of Aeshnidae in N-Germany. In the Zygoptera examined here, there is no such male avoidance strategy involving oviposition at lower TT to avoid MM. Here the beginning of oviposition and male flight activity is determined by the mating system.

Odonata Fauna of Istranca Mountains

In this study, Odonata fauna of Istranca Mountains has been investigated in spring, summer and autumn seasons of the years 1992-1993. During the research, 19 species belong to 11 genus of 4 families of suborder Anisoptera and 14 species belong to 10 genus of 5 families of suborder Zygoptera are determined from 39 different localities. Among these species, Pyrrhosoma nymphula (Sulzer, 1776) and Aeshna cyanea (Müller, 1764) are the new records for Turkish Thrace region. In examined habitats, Orthetrum brunneum, Ischnura elegans and Platycnemis pennipes are found to be the most common species. Orthetrum brunneum was observed to be located in 7, Ischnura elegans and Platycnemis pennipes were found to be located in 6 of 9 different habitats. These species oftenly observed around streams, irrigation canals, ponds, bushes and grass-type plants. The numbers of species were found only in one habitat are 13. The species established in our research area, zoo-geographically, represent different faunal elements namely 'Palaeogene", 'Entogene" and 'Exogene'. Species come from 'Entogene' group show very wide distribution in palearctic region and are very common in our research area.

Digestion of phosphatidylcholines, absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro.

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphatidylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1- [1-14C]acyl- and 1-[3H] alkyl-lysophosphatidylcholine, [1-14C]oleic acid, [2-14C]glycerol, phosphoryl[methyl-14C]choline, and [methyl-14C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A2, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and Pi, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoacylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine.

Is monoacylglycerol as an intermediate of triacylglycerol digestion absorbed by Aeshna cyanea larvae?

The isolated digestive juice of Aeshna cyanea larvae hydrolysed trioleoylglycerol preferentially at the terminal 1 and 3 positions, yielding 1,2-dioleoylglycerol as first intermediate. Hydrolysis continued to 1-and 2-monooleoylglycerol as second intermediate. Separate incubation of monooleoylglycerol revealed that hydrolysis could proceed to completion. Inadequate inhibition of mono[1- 14C]oleoylglycerol hydrolysis in the cold and in the presence of the lipase inhibitor tetrahydrolipstatin provided no information on whether monooleoylglycerol was absorbed in addition to free oleic acid. On the other hand, the analogue oleylglyceryl ethers were resistant towards hydrolysis by the digestive juice. Both monoethers and both diethers were esterified with [1- 14C]palmitic acid by the homogenate of the midgut wall, whereas esterification in vivo occurred only with the monoethers. These were recovered from the haemolymph after saponification of the joint diacylglycerol and acyl-0-alkylglycerol fraction, indicating that the monoethers had been absorbed and transported into the haemolymph. Ingestion of mono-1-0-[ 3H]octadecylglycerol showed that the ether was absorbed unchanged by the midgut epithelium, where the major part of the alkyl moiety was oxidized to free fatty acid and incorporated into phospholipids, acylglycerols and acyl-0-alkylglycerols. It is concluded from the absorption of the analogous monoalkylglyceryl ethers that monoacylglyceryl esters are also absorbed by Aeshna larvae.

Seasonal patterns of emergence in Odonata of a permanent stream in Southwestern Europe

By making regular, exhaustive, exuvial collections throughout the emergence period, the seasonal pattern of emergence was characterized for five species of Odonata in a permanent stream in southern Spain. Although emergence started at different times from mid‐April to late August, it was relatively well synchronised in all species, despite two (Cordulegaster boltonii and Onychogomphus uncatus) being ‘spring’ species (sensu Corbet, 1954) and the rest (Aeshna cyanea, Boyeria irene and Calopteryx haemorrhoidalis) being ‘summer’ species in the study habitat. Values for sex ratio at emergence are given.

Intestinal absorption of radioactive long-chain saturated fatty acids by the larval dragon-fly Aeshna cyanea and co-absorptive effects of other stable fatty acids

In contrast with unsaturated fatty acids, ingestions of long-chain saturated fatty acids by Aeshna cyanea larvae failed to induce an accumulation of lipid droplets in the intestinal absorptive cells, so that it remained questionable whether they were absorbed or not (Bauerfeind and Komnick, 1989). Therefore, we reinvestigated the question with the use of radioactive tracers, which were orally infused under two conditions: at low doses and at high doses prepared by the addition of other stable fatty acids. We found that carboxyl- 14C labelled long-chain stearic and palmitic acids, as well as the medium-chain lauric acid studied for comparison, were indeed absorbed from low doses of 7 nmol. They were incorporated predominantly into the phospholipids of the midgut wall and released into the haemolymph mainly as diacylglycerols. A substantial part was also transported as cholesterylester. The increase in total fatty acids of the infusates by the addition of 1.8 μmol stable oleic, arachidonic, myristoleic acids and lauric acid ethylester, which at high doses induce the formation of triacylglycerol droplets within the midgut enterocytes, markedly reduced the absorption rates of the radioactive saturated fatty acids and greatly favoured their incorporation into di- and triacylglycerols on the expense of the phospholipids and, at a minor part, on the expense of cholesterylester. By contrast, the addition of 1.8 μmol stable caproic acid yielded little, if any, effect on the absorption pattern of the radioactive palmitic acid. The results suggest that the co-absorption of medium- and long-chain saturated and unsaturated fatty acids is interdependent utilizing the same or similar uptake mechanisms and dose-dependent esterification pathways, while it is largely independent of and probably different from the absorption of short-chain fatty acids.

Assimilation and esterification of dietary fatty alcohol by the nymphal dragonfly, aeshna cyanea

(1) In vitro and in vivo studies on the midgut, haemolymph, fat body and Malpighian tubules fo Aeshna cyanea nymphs using [1- 14C]palmityl alcohol with and without unlabelled oleic acid revealed that free fatty alcohol was readily absorbed from the midgut lumen. (2) The absorbed fatty alcohol was partly assimilated in the midgut epithelium to fatty acid and esterified predominantly to polar lipids and acyl glycerols. (3) The non-assimilated alcohol was esterified with endogenous or dietary fatty acid to wax ester, which was released into the haemolymph and transported to the lipocytes of the fat body and Malpighian tubules. (4) Fatty alcohol assimilation was highest in the midgut wall and fat body, while wax ester synthesis was highest in the midgut lumen, followed by the midgut epithelium and fat body. (5) Dietary oleic acid reduced the concentration of free fatty alcohol as a result of pre-absorptive wax ester synthesis in the midgut lumen and decreased the rate of fatty alcohol absorption.

Intestinal absorption of cholesterol, transport in the haemolymph, and incorporation into the fat body and Malpighian tubules of the larval dragonfly Aeshna cyanea

Doses of 1.5 μ mol cholesteryl oleate ingested by Aeshna cyanea larvae were partially hydrolysed in the intestinal lumen and the liberated oleic acid absorbed, while free cholesterol and unhydrolysed cholesteryl oleate were eliminated in the faeces. Ingestion of [4- 14C]cholesterol dissolved in olive oil revealed that the larvae also absorbed free cholesterol, the minor part of which (up to 12%) was esterified in the midgut epithelium. Absorption and esterification were markedly enhanced when the same dose of labelled cholesterol was administered in 20% aqueous bovine serum albumin. Radioactivity was rather slowly released into the haemolymph where it was nearly exclusively associated with free cholesterol apart from traces of labelled cholesteryl ester which maximally amounted to 2.9% after 15 days. In the fat body labelled cholesteryl ester maximally amounted to 65% after 15 days, while in the triacylglycerol-storing Malpighian tubules it remained below 1% at all time periods investigated.

Dose-dependent incorporation of orally infused [1- 14C]oleic acid into the lipid classes of midgut, haemolymph and fat body of dragonfly larvae ( Aeshna cyanea)

1. 1. Five different doses of radioactive oleic acid (ranging from 1.87 nmoles to 5.61 μmoles) were administered to Aeshna cyanea larvae. 2. 2. Its incorporation into the midgut epithelium, haemolymph and fat body increased with the dose and time. 3. 3. Low doses caused up to 95% phospholipid labelling in the midgut wall, while labelled triacylglycerol was less than 1%, but increased with the doses to a maximum of 68%. The data favour the glycerophosphate pathway of oleic acid esterification. 4. 4. At low doses oleic acid was mainly released into the haemolymph from the midgut phospholipid pool, and at high doses from the triacylglycerol pool. 5. 5. Diacylglycerol was the most heavily labelled lipid class of the haemolymph, amounting up to 98% and slightly decreasing with time. 6. 6. The fat body showed a dose- and time-dependent increase in labelled phospholipid and triacyl-glycerol, maximally amounting to 14 and 90%, respectively.

Morphological specializations of dorsal rim ommatidia in the compound eye of dragonflies and damselfies (Odonata)

We have examined the fine structure of dorsal rim ommatidia in the compound eye of the three odonate species Sympetrum striolatum, Aeshna cyanea and Ischnura elegans. These ommatidia exhibit several specializations: (1) the rhabdoms are very short, (2) there is no rhabdomeric twist, and (3) the rhabdoms contain only two, orthogonally-arranged microvillar orientations. The dorsal rim ommatidia of several other insect species are known to be anatomically specialized in a similar way and to be responsible for polarization vision. We suggest that the dorsal rim area of the odonate compound eye plays a similar role in polarization vision. Since the Odonata are a primitive group of insects, the use of polarized skylight for navigation may have developed early in insect phylogeny.

Peroxisomal acyl-CoA oxidase and chain-shortening of dietary fatty acids in the midgut of dragonfly larvae, Aeshna cyanea

Activity of peroxisomal acyl-CoA oxidase was measured in homogenates of Aeshna cyanea midguts and found to be reversibly influenced by long-term fasting and refeeding. The enzyme was immunocytochemically colocalized with catalase in the peroxisomes of the intestinal absorptive cells. Orally administered nervonic and erucic acids were present in the triacylglycerol fraction of the midgut epithelium together with chain-shortened intermediates. But they were not detected in the diacylglycerol fraction of the haemolymph and triacylglycerol fraction of the fat body, which only contained chain-shortened intermediates. The prevalence of oleic acid in these three fractions suggests that the very long-chain fatty acids tested are assimilated by chain-shortening on their absorptive-pathway across the midgut epithelium.

Immunocytochemical localization of lipophorin in the fat body of dragonfly larvae ( Aeshna cyanea)

Immunocytochemistry using affinity-purified polyclonal antilipophorin revealed extracellular and intracellular locations of lipophorin in the fat body of dragonfly larvae. Lipophorin was associated with the external surface of the superficial and lateral plasma membranes which are directly exposed to the haemolymph. This location probably plays a role in the extracellular lipid-loading and unloading of lipophorin. Lipophorin was detected in the endoplasmic reticulum, Golgi apparatus and small vesicles located in the trans Golgi network region. These locations were interpreted as the secretory pathway of nascent lipophorin. Lipophorin was localized in various membrane-bound compartments which were identified as endosomes using biotinyl lipophorin and horseradish peroxidase as endocytotic markers. These results clearly suggested that circulating lipophorin was endocytosed by the adipocytes. Lipophorin endocytosis might serve the removal and lysosomal degradation of old lipophorin and/or provide an additional or alternative intracellular recycling mechanism of lipid-loading and unloading.

Lipid-loading and unloading of lipophorin in the midgut epithelium of dragonfly larvae ( Aeshna cyanea) : A biochemical and immunocytochemical study

Radioactive oleic acid orally infused into dragonfly larvae ( Aeshna cyanea) was recovered in the haemolymph in the esterified form as diacylglycerol which was transported by lipophorin. Radioactive diacylglycerol was released in vitro into the surrounding medium from the isolated, ligated midgut which had been preloaded in vivo with radiolabelled oleic acid. The release was largely dependent on the temperature and the presence of haemolymph in the incubation medium. On the other hand, the ligated midgut isolated from fasting larvae, was also able to absorb radioactive diacylglycerol from the haemolymph and to further esterify it to triacylglycerol. These results demonstrate that the midgut epithelium cannot only release but also absorb diacylglycerol at the basal side, which strongly suggests that both lipid-loading and unloading of lipophorin occur in the midgut probably in dependence of physiological conditions. Lipophorin penetrated across the basal lamina into the basolateral intercellular clefts of the midgut epithelium as evidenced by immunocytochemistry on the light and electron microscopic level, using affinity-purified polyclonal antibodies against lipophorin. The apicolateral intercellular clefts, i.e. the sites of the smooth septate junctions, and the intercellular clefts between the undifferentiated cells of the regeneration nidi remained unlabelled by antilipophorin. Incubation experiments with fluorescently labelled lipophorin suggested that lipophorin of the intercellular clefts was exchanged with lipophorin of the circulating haemolymph. Although the enterocytes showed clear endocytotic activity at the basolateral plasma membranes when incubated with horse radish peroxidase, lipophorin was not detected inside the cells by immunocytochemistry. Consequently, indications of lipophorin secretion and internalization were lacking. These results support the conclusion that lipid-loading and unloading of lipophorin occur extracellularly along the basolateral plasma membranes of the differentiated absorptive enterocytes.

The influence of tadpole swimming behaviour on predation by dragonfly nymphs

AbstractThis study analyses interspecific differences in anuran tadpole swimming behaviour that could influence vulnerability and therefore prey selection by dragonfly nymphs (Aeshna cyanea). Laboratory experiments showed that Bufo bufo tadpoles were almost continually and slowly in motion, which makes them easy prey for the dragonflies. Hyla arborea tadpoles were also very active and slow in swimming; in contrast to Bufo, however, they showed effective predator avoidance strategies (different habitat preferences, high evasiveness). The predation risk of Rana dalmatina larvae was low because the tadpoles were immobile and benthic, rare movements were made with high speeds. Bombina bombina is intermediate in swimming activity, velocity, and vulnerability.

Intestinal absorption of defined lipids by the larval dragonfly Aeshna cyanea (Insecta, Odonata): Wax esters and fatty alcohols

Dragonfly larvae are able to hydrolyse wax esters in the midgut lumen and to absorb both the fatty acid and the fatty alcohol moieties. The fatty acid is partly used for the synthesis of triglyceride and partly, together with the fatty alcohol, for the synthesis of wax ester. While the synthesis of triglyceride leads to the accumulative formation of lipid droplets in the midgut enterocytes, no accumulation of lipid droplets occurs after the ingestion of free fatty alcohol alone or of wax esters containing saturated long-chain fatty acids although in both cases wax ester is present in the midgut tissue. Under the same conditions wax ester is also present in the haemolymph, where it is associated with lipophorin. Time course studies suggest that the wax ester is transported from the midgut via the haemolymph to the fat body. The digestive juice and midgut wall are capable of acylating fatty alcohol when incubated in vitro in the presence of fatty acid. Wax ester is a natural constituent of the larval cuticle of the dragonfly Aeshna cyanea.

Peroxisomes of the midgut epithelium, malpighian tubules and fat body of larvae of the dragonfly, Aeshna cyanea

The enterocytes of the midgut epithelium of Aeshna cyanea larvae are rich in peroxisomes while the nidal regenerative and endocrine cells contain only a few. Most of the enterocytic peroxisomes are microperoxisomes lacking a crystalloid nucleoid, but peroxisomes with well developed nucleoid are also present. The peroxisomes are usually concentrated in the basal region of the cells but may also spread into the apical region and closely intermingle with absorptive lipid droplets. They significantly increase in number, when the larvae are regularly fed lipid-rich natural food or long-chain monounsaturated fatty acids that are unusual dietary components of these animals. This observation seems to indicate that the enterocytic peroxisomes are involved in chain shortening and degradation of fatty acids absorbed from the gut lumen. Numerous microperoxisomes are also present in the lipid-storing cells of the Malpighian tubules and fat body.

Intestinal absorption of defined lipids by the larval dragonfly Aeshna cyanea (Insecta: Odonata): Free and esterified saturated fatty acids

A total of 37 free and esterified fatty acids comprising 17 even- and odd-numbered saturates with chain lengths of C4–C22 were tested for their intestinal absorption by the larval dragonfly Aeshna cyanea, using the accumulation of lipid droplets in the midgut epithelium as a criterion for absorption. 1. (1) Short-chain saturates (C < 8) were more or less destructive to the midgut epithelium and did not lead to the formation and accumulation of lipid droplets. However, biochemical examinations after tricaproin ingestion revealed a time-dependent increase in the free fatty acid content of the midgut epithelium indicating that short-chain triglycerides were absorbed in the form of free fatty acids but did not lead to marked triglyceride synthesis in the absorptive cells. 2. (2) Individually and locally variable accumulations of lipid droplets were observed with even- and odd-numbered medium-chain C8 to C15 saturates regardless of whether they were administered as free or esterified fatty acids and regardless of their fluid or solid states. Examinations with thin-layer chromatography of experiments using esterified lauric acid revealed that glyceryl and ethyl esters were slowly hydrolysed in the midgut lumen and suggested that free fatty acid was the main absorbate, whereas the lipid droplets in the midgut epithelium predominantly consisted of triglyceride synthesized by the enterocytes from the absorbate. 3. (3) Absorption of long-chain saturates (C > 15) including palmitic acid which is the predominant saturated fatty acid of the natural diet remains questionable, because the epithelial lipid droplets, if present at all, were not markedly different from those also observed in starving controls. The results suggested that the absorption of saturates as assessed by the appearance of lipid droplets in the enterocytes is markedly inferior to the ready absorption of mono- and polyunsaturates previously demonstrated. They further suggest that Aeshna cyanea has a preference and two varied mechanisms for the absorption of short- and medium-chain saturates which, according to the literature, are only minor food and body constituents of insectivorous aquatic insects.

Development, cytology, lipid storage and motility of the Malpighian tubules of the nymphal dragonfly, Aeshna cyanea (Müller) (Odonata : Aeshnidae)

The Malpighian tubules of nymphal Aeshna cyanea (Odonata : Aeshnidae) were examined by light and electron microscopy. The 1st-instar nymphs have only 3 branchless tubules. With proceeding nymphal stages, these lengthen and branch. Also, additional tubules bud from the gut and show the same pattern of growth and branching, until in the final instar up to 21 separate tufts of branched tubules are present. A serpentine trachea/tracheole and a cross-striated muscle are helically wound around each tubule in close apposition. Isolated tubules show twisting movements for several days. Contraction of the muscle is responsible for fast coiling movements, while the slow decoiling movements probably depend on elastic deformations of the accompanying trachea, the basal lamina and the tubule cells, the latter showing an elaborate cytoskeleton and multiple adhesive junctions. The tubular epithelium consists of 4 types of cells. The distal segment is composed of ion transporting cells and terminates with a short, solid tip segment of undifferentiated cells. The intermediate segment consists of lipid cells which are densely filled with triglyceride droplets, as revealed by thin layer chromatography. Lipid cells are already present in the 1st instar before the nymphs have taken up any food. In later instars, the renal lipid content varies to some extent with the nutritional state and is nearly depleted during metamorphosis. The proximal segment is the region of tubular branching and may be conceived as the collecting duct of each tuft. Its epithelium consists of mucocytes.

Intestinal absorption of defined lipids by the larval dragonfly Aeshna cyanea (Insecta: Odonata): Mono- and polyunsaturated free fatty acids and their homotriglycerides

A total of 26 even-numbered unsaturates of chain lengths between C14 and C24 were screened for their enteric absorption by the larval dragonfly Aeshna cyanea. These included 14 cis- and 3 trans-monoenoics, 7 cis-polyenoics with up to 6 double bonds, and 2 trans-dienoics which were orally administered either as free fatty acids or/and as homotriglycerides. 1 and 2 days after ingestion the enterocytes contained more or less heavy accumulations of lipid droplets indicating that the tested unsaturates were readily absorbed by this species irrespective of chain length, number, position and configuration of double bonds. The equal utilization of free fatty acids and triglycerides, the digestion of trinervonin as tested by TLC, and the morphology of absorption suggested that the 12 different unsaturated triglycerides examined were taken up according to a common mechanism involving luminal lipolysis and cellular resynthesis.