Male Locusts Research Articles

Turnover of protein and diacylglycerol components of lipophorin in insect haemolymph

Lipophorin, radiolabelled in the protein or diacylglycerol moiety, was purified from adult locusts injected previously with [ 14C]protein hydrolysate or sodium[1- 14C]palmitate. The radiolabelled lipophorin was injected into adult male locusts and haemolymph samples taken periodically to determine the rate of disappearance of radioactivity from the haemolymph. Lipophorin was also purified from locusts that had been injected four days previously with ([ 14C]protein)-lipophorin to demonstrate that the radioactivity observed in the haemolymph at this time is due to radiolabelled lipophorin. The results indicate that the half-life of the protein component of lipophorin in resting insects is about 5–6 days whereas that of the diacylglycerol component is only about 2–3 hr. The results are consistent with the hypothesis that lipophorin functions as a “reusable shuttle” to transport a variety of lipid classes between sites of absorption, storage and utilisation.

Changes in haemolymph octopamine levels associated with food deprivation in the locust, Schistocerca gregaria

ABSTRACT. Levels of octopamine in the haemolymph and locomotor activity have been measured over 24 h in fed and food‐deprived adult male locusts, Schistocerca gregaria (Forskål). Locusts are predominantly diurnal, the locomotor activity of individuals with continuous access to food was higher during the photophase. The basal level of octopamine in haemolymph sampled during the first quarter of the light cycle was 7.8±1.1 pg/μl (51 nm). Depriving locusts of food at the start of the photophase for 9 h caused an increase in both the speed of movement and total activity compared to fed individuals. There was a corresponding increase in the haemolymph octopamine concentration which continued for as long as the insects were unable to feed, reaching a concentration of 20.7 ± 3.7 pg/μl (135 nM). While declining, the levels of octopamine remained higher in fed locusts for up to 4 h after replacing the food. The results are discussed in relation to the regulation of feeding behaviour and a proposed neurohormonal role of octopamine in insects.

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts.

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.

Le dimorphisme sexuel des protéines cuticulaires du Criquet migrateur, Locusta migratoria migratorioides (Reiche &Farmaire)

Cuticular and blood proteins were studied in adult male and female Locusta migratoria migratorioides (Reiche &Farmaire) by means of electrophoresis and imunoelectrophoresis. Comparative analysis of proteinograms, physico-chemical characteristics (Rf, molecular weight), and antigenic properties allow us to conclude that some cuticular proteins are sex related; this sexual dimorphism is correlated with the maturation process; one of the specific female cuticular proteins is closely related to vitellogenin. Many of the cuticular proteins exhibit the same electrophoretic pattern and the same antigenic determinants as blood proteins which appear to pass the epidermis without any major structural alteration. Other ones are typically cuticular and are presumably synthetized by epidermal cells using blood precursors. There are various major hemolymph proteins which are never detected in the cuticle; this suggests that epidermal cells recognize and select, among the blood proteins, those which are incorporated into the cuticular matrix during the moulting process and oocyte maturation.

X-linkage of a vitellogenin gene in Locusta migratoria

Hybridization of a cloned DNA probe to blots of restriction digests of Locusta migratoria genomic DNA showed that a vitellogenin gene is present at one copy per haploid genome in females, and at only one-half that number in males. The genomic region encompassing the 3′-coding end of the gene is polymorphic, as revealed by blots of DNA from individual locusts. DNA from some female locusts yielded two variants in this region, whereas a series of male locusts showed one variant or the other, but never both. The results demonstrate that the vitellogenin gene, which is normally expressed only in females, is X-linked in L. migratoria.

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ⪢ adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.

The effects of precocene II on early adult development in male Locusta

Haemolymph protein synthesis and the accumulation of protein in haemolymph and accessory reproductive gland of normal adult male locusts were compared to that of males treated with precocene II. The precocene-treated insects showed a lower level of protein synthesis in both the fat body (source of haemolymph protein) and accessory reproductive gland. Topical application of juvenile hormone reversed the effects of precocene treatment. It would appear that the observed effects are the result of a reduction in juvenile hormone titre in the treated animals.

Effects of flight training on flight speed and substrate utilization in locusts

ABSTRACT. Regular flight exercise of adult male Locusta migratoria migratorioides (R and F) accelerated the development of maximum flight speed and disrupted the development of the typical pattern of change of flight speed exhibited when normal (untrained) adult male laboratory locusts are flown on roundabouts. Thus, while untrained mature locusts fly fast initially and then slow to a steady cruising speed after 20 min, trained locusts flew at a relatively constant speed throughout a 60‐min test period. Flight training also led to a marked reduction in the size of the fat body and the flight muscles, but flight muscle ultrastructural development was not affected. Regular flight exercise had no long‐term effect on haemolymph carbohydrate concentration but lipid levels were significantly depressed.

Endocrine control of hemolymph carbohydrates in Locusta migratoria: Comparison between effects of two endogenous hormonal extracts and effects of insulin and glucagon

1. 1. In adult male Locusta, effects of corpora cardiaca extracts, intestinal insulin-like molecule, glucagon and insulin were tested on hemolymph carbohydrates. 2. 2. Carbohydrates were separated by gas-liquid chromatography. 3. 3. CC extracts and glucagon mainly increase trehalose and melibiose levels; they have no effect upon glucose concentration. 4. 4. Intestinal insulin-like molecule and insulin decrease all hemolymph carbohydrates. 5. 5. These results suggest that in Locusta, like in mammals, hemolymph carbohydrates are controlled by a double system of regulation: a hyperglycemic system present in CC and a hypoglycemic factor localized in midgut.

Interconversions of diacylglycerol-transporting lipoproteins in the haemolymph of Locusta migratoria

The diacylglycerol-carrying lipoproteins in the haemolymph of adult male Locusta migratoria appear to be dynamic structures which participate in exchange and transfer processes involving their lipid and protein moieties. Between the two lipoprotein fractions present in the resting condition (O and A yellow), a rapid exchange of apoprotein is observed upon re-injection of isolated and [ 125I]-labelled lipoprotein A yellow. Furthermore, a high exchange rate of diacylglycerol is inferred from the incorporation of [ 14C]-labelled diacylglycerol into the lipid components. Elevation of the haemolymph diacylglycerol level in response to injection of adipokinetic hormone is accompanied by the appearance of a high molecular weight lipoprotein A + and an increase in lipoprotein O, originating from a transfer of apoprotein from [ 125I]-A yellow to both lipoproteins. Simultaneously, radioiodinated non-lipid containing haemolymph protein C participates in their formation. In contrast, [ 125I]-protein B is not involved in the lipoprotein remodelling. Though the increased diacylglycerol moieties carried by A + and O are derived principally from the augmented release of lipid from the fat body, net transfer of [ 14C]-diacylglycerol from A yellow to both higher molecular weight lipoproteins evidences participation of the diacylglycerol component as well.

Immunological relationships between the diacylglycerol-transporting lipoproteins in the haemolymph of Locusta

Abstract 1. 1. Immunoelectrophoresis and immunodiffusion of the diacylglycerol-carrying protein fractions isolated by gel filtration from the haemolymph of male locusts at rest and after injection of the adipokinetic hormone evidenced an immunological relationship between all lipoprotein fractions. 2. 2. The two lipoprotein fractions (A yR and O R ) occurring in Locusta at rest are immunologically identical; the very high molecular weight fraction O R apparently is a polymer of basic lipoprotein A yR . 3. 3. In the lipoprotein fraction A + appearing after injection of the adipokinetic hormone, as well as in the elevated fraction O AKH , two protein constituents are immunologically identical with the basic lipoprotein A yR . In addition, the third protein component is descended from the non-lipid carrying haemolymph protein C.

Neurosecretory protein synthesis in relation to starvation and feeding in adult male Locusta migratoria

An immunological approach has been used to assess the effects of starvation and subsequent feeding upon the synthesis of neurosecretory protein in adult male Locusta migratoria. Starvation (for 5 days) did not reduce the incorporation of radioactive cystine into neurosecretory protein. However, the rate of neurosecretory transport in starved insects was approximately half that in normal fed insects. Within 1 hr after the start of feeding the rate of neurosecretory protein synthesis more than doubled and the rate at which newly synthesized protein was transported to the CC increased approximately three-fold. The incorporation of cystine into specific (i.e. neurosecretory) protein was always higher, even in starved insects, than into the non-specific proteins (i.e. proteins other than those from the A-cells) of the brain. This difference was maximal at 4 hr after feeding when the incorporation into specific protein was more than 5 times that into non-specific protein.

Locust collagen: morphological and biochemical characterization.

Segment-long-spacing crystallites and reconstituted fibrils have been made from collagen extracted from the ejaculatory duct of the adult male locust, Locusta migratoria. These show the same banding pattern after positive staining as segment-long-spacing crystallites and fibrils made from mammalian type I collagen. Native fibrils show the same periodic pattern as type I fibrils, but it is not so distinct. Biochemical analysis of pepsin-digested locust collagen shows that there are two collagenous components. The alpha chains of the major component are similar to mammalian alpha 1 (I) chains, except that the number of hydroxylysine residues is elevated and the CNBr peptides differ. There are no alpha 2 chains; hence this locust collagen molecule is an alpha 1 trimer. The second component, which is present in only minute quantities, may be a type IV basement membrane collagen. It is concluded that the fibrous collagen molecule of the locust is very similar to that of mammalian type I trimers and to those of other invertebrates which have been examined.

Studies on l‐glutamate in insect haemolymph.

ABSTRACT. Glutamate was detected in the haemoplasm of adult male Periplaneta americana, adult male Schistocerca gregaria, adult male Locusta migratoria, larval Calliphora erythrocephala, larval Lucilia sericata and Carcinus maenas, in all cases at low concentrations only, i.e. below 10‐5M. This was confirmed by high‐voltage paper electrophoresis and, in the case of Lucilia sericata larvae, by the lack of pharmacological activity of the haemolymph. Glutamate was detected in appreciable concentrations in the haemocytes of all the arthropods analysed. It was found that levels of glutamine in the haemoplasm increased if the haemolymph had started to clot, if the haemolymph had been inadequately centrifuged, if the haemolymph was contaminated by other tissues and if acid protein precipitants were used which hydrolysed glutamine to glutamate. This finding removes a major argument against the acceptance of l‐glutamate as a neuromuscular transmitter.

Studies on l‐glutamate in insect haemolymph.

ABSTRACT. l‐Glutamate when injected into the haemolymph of Lucilia sericata larvae and adult male Locusta migratoria was rapidly removed by uptake mechanisms to other tissues in the insect. Data from Lucilia larvae indicate that following uptake glutamate is metabolized and the metabolites are secreted back into the haemolymph. l‐Aspartate injected into the haemolymph of Lucilia larvae was also rapidly removed. When both l‐aspartate and l‐glutamate were injected simultaneously, the rate of glutamate removal was significantly reduced. It is concluded that glutamate and aspartate share the same uptake mechanisms. l‐Leucine injected into Lucilia larvae and Locusta was removed at a significantly slower rate than glutamate or aspartate.

Studies on l‐glutamate in insect haemolymph.

ABSTRACT. The neuromuscular junctions of abdominal body wall muscles of larval Lucilia sericata are mainly superficial, with no glial sheath, and are surrounded by a well‐developed subsynaptic reticulum. Injection of l‐glutamate into the haemolymph of adult male Locusta migratoria and larvae of Lucilia sericata caused reversible effects on motor activity. These effects were quantified and dose—response curves are presented. The effective concentration of the injected doses is low enough to be physiologically relevant. Injection of l‐aspartate into Locusta haemolymph affected motor activity. Injection of l‐aspartate into Lucilia larvae produced no detectable effect. Injections combining l‐aspartate and l‐glutamate into Lucilia larvae had a synergistic effect on the duration of paralysis, compared with the same concentration of l‐glutamate injected alone.

SLS crystallites and reconstituted fibrils made from locust collagen

That insects possess collagen fibrils similar to those of vertebrates is well established. In some insects, the fibrils are very thin and indistinctly banded, while in others, for example, locusts and cockroaches, fibrils of larger diameter and distinct banding are found. Estimates of the periodicity of the banding of sectioned fibrils range from 56nm to 63nm (see 2), but the interband patterns are similar; the variation in the periodicity is probably due to the preparative procedures. A peculiarity of many of the fibrils is that on ageing they become very large and irregular in cross-section; typical examples are found in the connective tissue around the ejaculatory duct of 2-3 week old adult male locusts, Locusta migratoria. Similar large, irregular fibrils are found in some pathological conditions in mammals. One way to establish similarities between collagens is to compare their segment-long-spacing (SLS) crystallites and reconstituted fibrils, and so these experiments were performed with locust collagen.Locust collagen is not readily soluble in salt or dilute acids, and so to increase its solubility, newly-moulted, adult male locusts were fed on wheat seedlings sprayed with a 1% solution of β-aminoproprionitrile, a lathrytic agent which reduces cross-linking in mammalian collagens.

Dynamics in the haemolymph trehalose pool during flight of the locust, Locusta migratoria

In adult male Locusta changes in level and specific radioactivity of the haemolymph trehalose pool were studied during flight and rest after pulse-labelling with [U- 14C]-trehalose. In the initial period of flight, consumption of haemolymph trehalose is high (about 7.3 mg/animal/hr), but there is little mobilization of trehalose from body carbohydrate stores. After 30 min of flight, however, steady state conditions in the haemolymph trehalose pool are reached in which the turnover rate of the (constant) pool amounts to 2.4 mg/animal/hr. At rest, turnover rate of the haemolymph trehalose pool is significantly lower (0.5 mg/animal/hr). It is concluded that during sustained tethered flight (when diglyceride is the predominant energy source) trehalose contributes substantially to flight muscle energy metabolism since 23% of the energy supply originates from carbohydrate oxidation.

Turnover of locust haemolymph diglycerides during flight and rest

The constant, elevated diglyceride concentration in the haemolymph of adult male Locusta migratoria during sustained tethered flight (14.1 ± 1.2 mg/ml haemolymph) was established to be a steady state, in which the rate of diglyceride mobilization matched the rate of utilization. Pulse-labelling of the haemolymph diglycerides with [1- 14C]-oleic acid in steady state flight conditions results in a turnover time of about 1 hr for the diglyceride pool, whereas in animals rested after flight values close to 8 hr were obtained. So, during flight there is a sharp increase in lipid transport, the initial turnover rate in the resting animal (0.3–0.5 mg diglyceride/hr) reaching 3.4 mg diglyceride/hr at the steady state flight level. As identical turnover rates were obtained using [1- 14C]-palmitic acid or [1- 14C]-linolenic acid, apparently there is no preferential utilization of diglycerides with a specific fatty acid composition in flight muscle energy metabolism. The characteristic diglyceride fatty acid spectrum at the steady state flight level remained remarkably constant during flight performances lasting for 6 hr. The above findings indicate homogeneity of the haemolymph diglyceride pool and suggest random utilization of fatty acids.

The energetics of the jump of the locust Schistocerca gregaria.

The anatomy of the metathoracic leg is redescribed with particular reference to storage of energy in cuticular elements and the way in which the stored energy is used in jumping. The jump of adult male locusts requires an energy of 9 mJ and that of the female requires 11 mJ. The semilunar processes of each metafemur store 4 mJ at a stress of 15 N, and the extensor tibiae apodeme stores a further 3 mJ at the same stress. The total stored energy in both metathoracic legs is 14 mJ. The extensor tibiae muscle produces a maximum isometric force of over 15 N at 30 degrees C and, when loaded with the extensor apodeme and semilunar processes, attains this force in 0.3 sec with a strain of 0.8 mm. The peak power output is 36 mW or 0.45 W.g-1. The peak isometric force is attained when the tibia is fully flexed and the force falls as the tibia extends. The extensor tibiae muscle A band is 5.5 mum long and the peak force is over 0.75 N.m-2. The peak velocity of shortening is 7 mm.sec-1 or about 1.75 lengths/sec at 30 degrees C. The tensile strength of the extensor apodeme is 0.6 kN.mm-2 and Young's modulus is 19 kN.mm-2. The safety factor does not exceed 1.2 and the safety factor of the semilunar processes and tibial cuticle is little higher. The jump impulse lasts 25-30 msec. A velocity of 3.2 m.sec-1 is reached after a peak acceleration of 180 m.sec-2. The peak power output is 0.75 W at close to maximum velocity. Energy losses in rotating the femur and tibia are small and it is shown that the leg is able to extend at 7 times the normal rate with losses of about 20%. Most of the stored energy is converted to kinetic energy as the animal jumps. A model is based on the relaxation of a spring that has the properties of the elastic elements of the locust leg into a lever with the same kinematics as the locust leg produces a force-distance curve similar to that measured for locust jumps. The major part of the jump energy is stored before the jump.