Transport of lipids in insects

Many insect species are almost completely dependent on lipids for their metabolic needs, although this is usually a function of developmental stage. The primary storage organ is the fat body, which can constitute 50% of the fresh weight of the insect and also acts as the major metabolic center (analogous to the vertebrate adipose tissue and liver). Bathing the fat body (and all other tissues and organs) is the hemolymph, the main functions of which are to transport nutrient substrates to utilization sites and to deliver metabolic wastes to the excretory system. Although neutral lipids are stored as triglycerides, in times of need they appear to be endergonically released into the hemolymph as diglycerides in the majority of insects thus far studied (particularly silkmoths and locusts). Indeed, diglycerides constitute the largest neutral lipid fraction in the hemolymph of silkmoths, locusts, cockroaches, bugs, etc. In the hemolymph the diglyceride is found as a constituent of specific lipoproteins, and one specific lipoprotein class (lipoprotein I; high density lipoprotein) appears to be necessary for the transport of diglyceride from the fat body cell into the hemolymph. This particular lipoprotein is also involved in the transport of cholesterol from the gut into the hemolymph. Thus, lipoprotein I appears to be the major neutral lipid and sterol transport agent in the insects studied and, in addition, plays a regulatory role in the release of both diglycerides and sterols. Hemolymph lipoprotein II (very high density lipoprotein) may be important in providing protein and lipid to the insect ovary during oogenesis. Ecdysone, the polyhydroxy steroidal insect molting hormone, is probably carried "free" in the hemolymph, although reports exist of specific hemolymph-binding proteins in some species. The other major insect growth hormone, juvenile hormone, is transported by hemolymph lipoproteins in silkmoths and locusts and by a lower molecular weight hemolymph protein in the tobacco hornworm.     

Receptor-mediated endocytosis of lipid and lipophorin by the larval fat body, adult ovary and testis in the wax moth Galleria mellonella

Lipophorin (Lp) in the hemolymph of insects is known to selectively deliver lipids from sites of absorption or synthesis to sites of storage and utilization, such as the fat body, ovary and testis; however, no study regarding this has been reported in Galleria mellonella. In the present study, we examined the process by which Lp is taken up into the larval fat body, adult ovary and adult testis, and the transfer of lipid by Lp to these tissues in Galleria mellonella. To investigate the involvement of a receptor in Lp endocytosis, the larval fat body, adult ovary and adult testis were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)‐Lp, FITC‐Lp plus unlabeled Lp, and FITC‐Lp plus suramin, a receptor endocytic inhibitor. The amounts of FITC‐Lp in the three tissues were significantly decreased in the presence of unlabeled Lp and suramin, indicating that endocytosis of Lp by the tissues is mediated by a receptor. To examine the transfer of lipid by Lp, the tissues were incubated for 1 h at room temperature with 1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI)‐Lp, DiI‐Lp plus unlabeled Lp, and DiI‐Lp plus suramin. The transfer of lipid by Lp was inhibited in the presence of unlabeled Lp and suramin, which is consistent with a receptor‐mediated process. Our results show that the transfer process of lipid by Lp and uptake of Lp itself is by receptor‐mediated endocytosis.     

Characterization of termite lipophorin and its involvement in hydrocarbon transport

The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae.     

Lipophorin uptake by the larval fat body and adult ovary in the wax moth Galleria mellonella

Lipophorin (Lp) acts in the circulation of insects to selectively deliver lipids to target tissues. In the present study, we wanted to show that Lp is taken up into larval fat body cells and the adult ovary in Galleria mellonella. Larval fat body and adult ovary tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)‐labeled Lp. Fluorescence microscopy and sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) revealed that fat body and ovary tissues internalize fluorescence‐labeled Lp. The results suggest that both lipids and proteins are taken up by fat body cells and the ovary and also that large amounts of proteins and lipids taken up can serve as building blocks and as a source of energy. Immunological relationships with other insects were investigated using western blotting. The data showed that the Lp of Galleria mellonella is related to that of Hyphantria cunea.     

Metamorphosis of a protein

All insects appear to have a transport lipoprotein in the hemolymph (blood) that is responsible for moving hydrophobic materials through aqueous compartments. This has been called lipophorin because it is believed to be a reversible transport shuttle. Since most insects undergo some degree of metamorphosis from larval stages to the adult, the need to transport hydrophobic materials or the nature of these materials may change in the course of the life span. This is especially marked in the case of the holometabolous insects – those which undergo drastic change of form, e.g. caterpillar-pupa-adult moth. We have found that lipophorin undergoes a molecular metamorphosis paralleling that of the insect.     

Site of synthesis,tissue distribution,and lipophorin transport of hydrocarbons in Blattella germanica (L.) nymphs

The site of hydrocarbon (HC) synthesis and the amount of HC in various tissues were investigated in relation to developmental stage in the last larval stadium of the German cockroach, Blattella germanica. Abdominal integument linearly incorporated [1-(14)C]propionate into HC for at least 6h in vitro, whereas other body parts synthesized little or no HC. The third through sixth abdominal sternites and tergites were the principal sites of synthesis. High rates of HC synthesis resulted in a fivefold increase in internal HC during the last stadium. We examined the distribution of HC in the hemolymph, fat body, and the developing imaginal cuticle. Hemolymph HC titer was relatively constant at approximately 8&mgr;g/&mgr;l. However, as hemolymph volume increased from 5 to 11&mgr;l in the first 4days of the last stadium, HC content increased and then remained stable the remainder of the stadium. Lipophorin, immunoprecipitated with adult lipophorin polyclonal antibodies, was the only HC carrier protein in nymphal hemolymph and its HC profile was identical to that of hemolymph and similar to that of the epicuticle. The concentration and total amount of hemolymph lipophorin increased until 3days before adult eclosion and declined immediately after ecdysis. The HC content of non-biosynthetic integument (legs, pronotum) doubled during formation of the imaginal cuticle, as did the HC content of sternites, which synthesize HC. HC content of fat body, however, increased threefold during the same period, suggesting that the fat body serves as a storage site for HC during cuticle formation. We conclude that in the last stadium HC is synthesized by abdominal oenocytes, loaded onto hemolymph lipophorin, and transported to fat body and both nymphal and imaginal cuticle. Hydrocarbons associate with the imaginal integument several days before eclosion.     

EFFECT OF STARVATION ON LIPOPHORIN DENSITY IN FIFTH INSTAR LARVAL Manduca sexta

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2–3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp‐larval₁, HDLp‐L₁) and 1.141 ± 0.002 g/ml (HDLp‐L₂). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp‐L_s). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp‐L_s was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24‐h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24‐h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.     

Regulation of JH titers: The relevance of degradative enzymes and binding proteins

Juvenile hormones play a crucial role in development, metamorphosis, and reproduction of insects. This mini-review discusses the nature of the juvenile hormones identified in insects and their changes in concentration in the hemolymph during development and reproduction. The hemolymph titer is largely determined by the rate at which juvenile hormones are synthesized and released by the corpora allata, but other factors are also involved in titer regulation, such as the affinity and concentration of juvenile hormone binding proteins in the hemolymph and the rate of juvenile hormone degradation in hemolymph and tissues. Juvenile hormone specific esterases occur in hemolymph and tissues, whereas epoxide hydrolases, which may degrade the hormone, are exclusively tissue bound. The activities of these degradative enzymes and the concentration of binding proteins change during the insect life cycle and these changes are related to fluctuations in hormone titer. However, we are still a long way from understanding the subtle interactions between these components in regulation of juvenile hormone titers. In particular, our knowledge is hampered by lack of information about the types, concentrations, and affinities of intracellular juvenile hormone receptors. © 1996 Wiley-Liss, Inc.     

A novel lipoprotein from the hemolymph of the cochineal insect, Dactylopius confusus.

A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.     

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp ⁽¹²⁵I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when ¹²⁵I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a K_dof 7.1 ± 0.9 x 10^-8M and a maximal binding capacity of 430 ± 40 ng ¹²⁵I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.     

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.     

Blue lipophorin from Podisus maculiventris

A chromoprotein responsible for the blue coloration of the hemolymph in the spined soldier bug, Podisus maculiventris (Say), was isolated and identified as lipophorin. With the exception of its blue color the lipoprotein shares similar molecular characteristics with the hemolymph lipophorins of other Hemipterans and insects of several different orders. Its ability to carry a blue chromophore, biliverdin IX γ, adds a new feature to this multifunctional lipoprotein. © 1992 Wiley-Liss, Inc.     

Absorption and storage of phosphorus by larval Manduca sexta

The role of phosphorus (P) in numerous important biological structures, coupled with the observation that P-content of many insect foods is disproportionately low, suggests that P may be a critical nutrient for growing insects — however, the few studies examining the effects of dietary P on insect performance have generally found only weak relationships. This mismatch may be reconciled by understanding the physiological mechanisms by which insects handle P. Here we describe P processing by larvae of Manduca sexta. When given un-manipulated leaves of a common host plant, Datura wrightii, fifth-instar larvae retained about 85% of P consumed; when given P-enriched leaves larvae retained only 25% of P consumed. Analysis of gut concentrations of P at four sites along the digestive tract, and in leaves and feces, indicates that the rectum is the primary site of P transport between the gut and body and that differences in P retention may be accounted for by differential rates of rectal P transport. Larvae given P-enriched leaves also showed an eightfold increase in the concentration of P in the hemolymph, primarily as α-glycerophosphate — but only a 12% increase in the concentration of P in body tissues, suggesting that hemolymph plays a central role in storage and buffering of P.     

Flight metabolism in Panstrongylus megistus (Hemiptera: Reduviidae): the role of carbohydrates and lipids

The metabolism of lipids and carbohydrates related to flight activity in Panstrongylus megistus was investigated. Insects were subjected to different times of flight under laboratory conditions and changes in total lipids, lipophorin density and carbohydrates were followed in the hemolymph. Lipids and glycogen were also assayed in fat body and flight muscle. In resting insects, hemolymph lipids averaged 3.4 mg/ml and significantly increased after 45 min of flight (8.8 mg/ml, P < 0.001). High-density lipophorin was the sole lipoprotein observed in resting animals. A second fraction with lower density corresponding to low-density lipophorin appeared in insects subjected to flight. Particles from both fractions showed significant differences in diacylglycerol content and size. In resting insects, carbohydrate levels averaged 0.52 mg/ml. They sharply declined more than twofold after 15 min of flight, being undetectable in hemolymph of insects flown for 45 min. Lipid and glycogen from fat body and flight muscle decreased significantly after 45 min of flight. Taken together, the results indicate that P. megistus uses carbohydrates during the initiation of the flight after which, switching fuel for flight from carbohydrates to lipids.     

Changes of the Antioxidant Status and System of Generation of Free Radicals in Hemolymph of Galleria mellonella Larvae at Microsporidiosis

Changes of superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities as well as of the content of SH-containing compounds were revealed in hemolymph of the native and the Vairimorpha ephestiae microsporidian-infected greater wax moth Galleria mellonella larvae. The SOD and GST activities in hemolymph of infected insects decreased at the stage of merogony, whereas during massive sporulation the enzymatic antioxidant activity in host tissues was higher than in control. By the ESR spectroscopy method, using the 1-hydroxy-3-carboxy-pyrrolidinespin-trap, generation of free radicals in hemolymph of infected insects was shown to decrease only at the stage of sporogony. The phenoloxidase activity in lymph was lower at acute microsporidiosis than in native larvae. The hemolymph concentration of thiol-containing proteins in infected insects did not differ from that in control. We suggest that decrease of generation of free radicals in hemolymph of the greater wax moth larvae at the stage of sporogony is due to a suppression of the prophenoloxidase system and an elevation of the antioxidant activity.     

Transport and utilization of free fatty acids in Triatoma infestans

Triatoma infestans hemolymph has 0.31 mg/ml of free fatty acids and 2.8 mg/ml of diacylglycerols. Almost all the diacylglycerols are transported by lipophorin whereas free fatty acids are carried by lipophorin and a very high density lipoprotein. The binding of cis-parinaric acid to lipophorin was employed to specify the free fatty acid binding properties of lipophorin. Lipophorin has 10 binding sites of high affinity (3 x 10(7)) and approximately 45 binding sites of low affinity (1 x 10(6)). The relative rate of tissue incorporation of free fatty acids and diacylglycerols was measured by injecting insects with hemolymph previously labeled in both, free fatty acids and diacylglycerols. In this way, the half-life of the hemolymph free fatty acids was estimated to be about 4 min. Based on this result and taking into account the content of free fatty acids and diacylglycerols in hemolymph, the incorporation of free fatty acids, expressed in moles of fatty acids, seems to be 3.4 times higher than that of diacylglycerols. This finding can be applied to other insects.     

Wound Responses in Insects

For over a century, the study of specific antipathogenic strategiesin insects has been confounded by non-specific responses tointegumental invasion. Experimental injury to diapausing Hyalophoracecropia silkmoth pupae elucidated some of the events inherentin this response—increased oxygen consumption and DNAand RNA synthesis leading to de novo synthesis of proteins,some of which are constituents of the adult protein cohort aswell as some injury-specific ones. The mechanism which enforcesdiapause is apparently released by integumental injury as wellas by normal developmental stimuli. Recent work has concentratedon purification of antipathogenic and injury-specific proteins,the possible involvement of lectins in the immune response,and localization of synthesis of these proteins in hemocytesand fat body cells. At least ten different hemolymph proteinswhich are synthesized by fat body cells in response to inoculationof lepidopteran species with bacteria currently are being isolated.The hemolymph of H. cecropia contains lectins which are synthesizedby hemocytes. Analysis of in vitro incorporation of ['H]leucineby hemocytes into proteins reveals that these lectins apparentlyare not constituents of the secreted injury response proteincomplex in fifth instar caterpillars or diapausing pupae, norare hemolymph lectin titers significantly different in healthyversus diseased or injured animals. However, intracellular lectinconcentrations may increase upon injury. Increased lectin titerand induction of bactericidal activity coincide in another holometabolousspecies, the fleshfly Sarcophaga peregrina. Pursuit of thesestudies may elaborate our knowledge of insect cellular immunity.     

Lipophorin of the larval honeybee, Apis mellifera L

Most insects have a major lipoprotein species in the blood (hemolymph) that serves to transport fat from the midgut to the storage depots in fat body cells and from the fat body to peripheral tissues. The generic name lipophorin is used for this lipoprotein. In larvae of the honeybee, Apis mellifera, a lipophorin has been found with properties that correlate well with those of the only other lipophorin reported for an immature insect, that of the tobacco hornworm, Manduca sexta. The honeybee lipophorin (Mr = 530,000) has a density of 1.13 g/ml, contains approximately 41% lipid and 59% protein, and contains two apoproteins, apoLp-I, Mr = 250,000 and apoLp-II, Mr = 80,000, both of which are glycosylated. The lipids consist predominantly of polar lipids, of which phospholipids and diacylglycerols represent 60% of the total. When the intact lipophorin is treated with trypsin, apoLp-I is rapidly proteolyzed, while apoLp-II is resistant, indicating a difference in exposure of the two apoproteins to the aqueous environment. Honeybee apoLp-II cross-reacts with antibodies to M. sexta apoLp-II, but not to anti-M. sexta apoLp-I. No cross-reactivity of honeybee apoLp-I to anti-M. sexta apoLp-I was observed.