Development of the subsoephageal body cells and the pericardiac cells during embryogenesis with diapause in Locusta migratoria (Linnaeus 1758) (Orthoptera: Acrididae)

During Locusta migratoria embryogenesis, the yolk is progressively degraded and the resulting metabolites are released in the haemolymph. We researched the organs possibly involved in the uptake of haemolymphatic proteins. Among organs originated from mesoderm, the SOB (suboesophageal bodies) situated in the embryonic head are remarkable by a very early acquisition of differentiated cytological characters, while most other cells of the embryo are undifferentiated. The SOB quite disappear before hatching. Just before katatrepsis stage, the other organs derived from mesoderm begin to differentiate, including the PC (pericardial cells) which take over from the SOB. These cells, situated in thorax and abdomen, are developed during the dorsal close of embryo. The development and the ultrastructural changes of the SOB cells and of the PC were studied during an embryogenesis with diapause. The morphology of embryos which enter diapause is comparable with that of a continuous development at the beginning of katatrepsis. However, the cells of SOB and PC cells suffer from remarkable changes not only physiologically but cytologically. At the beginning of diapause, the proteosynthetic activity practically disappears in the SOB cells and the lysis areas appear. Nevertheless, the exchanges between these cells and the haemolymph still remain important. For the period of cold, which is necessary to the resumption of development, the aspect of the SOB cells changes and in particular the areas of lysis become less wide. When the embryo reopens its development, the SOB cells show a proteosynthetic activity and the areas of lysis disappear. The changes of the SOB cells and of the PC cells are regularized during the resumption of the development: the SOB cells which had again taken a normal activity start to regress from the stage VII on, while the PC cells take over.     

The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Trichoplusia ni

Brain (median or lateral regions) or suboesophageal ganglion (SOG) homogenates of Day 1 fifth instar larvae of Trichoplusia ni induced the appearance of haemolymph juvenile hormone esterase (JHE) when injected into Day 1, Day 2 or early Day 4 fifth instar ligated hosts. Brain and SOG homogenates of late fourth instars also induced JHE when injected into Day 1 hosts, whole late fifth instar and pupal tissue did not. The pattern of JHE induction by early fourth through Day 3 fifth instar brain and SOG homogenates correlated with natural haemolymph JHE activity occurring at these times. Implantation of late fourth and Day 1 fifth instar brains and/or SOG into similar age hosts similarly induced JHE activity while prothoracic and abdominal ganglia did not. The relative levels of induction following implantation were SOG<brain<brain+SOG. JHE activity which appears in the haemolymph following injection of brain homogenates appears to be largely due to a single enzyme which has an isoelectric point indistinguishable from that of the natural haemolymph enzyme. Evidence is presented which suggests that inhibitory as well as stimulatory brain factors are involved in JHE regulation.     

Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects

When an extract of the corpora cardiaca/corpora allata from two species of wingless stick insects, Carausius morosus and Cuniculina impigra, which cause no adipokinetic or hyperglycaemic effect when injected into the donor insects themselves, is injected into adult Locusta migratoria it resulted in an increase in the haemolymph lipid concentration. The lipid elevation was time dependent, with a maximum effect about 90–180 min after injection, and was also dose-dependent. About 0.001–0.002 (C. morosus) and 0.01 (C. impigra) gland equivalents were needed to produce a significant increase; a maximal effect was reached with approx. 0.075 (C. morosus) and 0.25 (C. impigra) gland equivalents. Carausius extract was also able to elevate carbohydrate concentration in the haemolymph of Periplaneta americana. However, the effect was weak and no maximal response was reached even with a dose of 0.5 gland equivalents. Adipokinetic hormone activity was present in CC/CA extracts of larval Carausius; the activity was about 30 times lower in 1-day-old 2nd instar individuals, and approx. 5 times less at the beginning of the 6th instar than that found in adults. In both stages the hormone levels increased gradually from the beginning to the end of the instar. No age-related changes were observed during the adult stage. Further studies on the lipid-mobilising factor of C. morosus revealed that it was stored entirely in the CC and not in other nervous tissue, e.g. brain, CA, suboesophageal ganglion, thoracic and abdominal cord. The factor was heat stable for at least 1 hr at 100°C and retained its adipokinetic activity after incubation with trypsin and the exopeptidases such as carboxypeptidase A and leucine aminopeptidase. However, activity was abolished when incubated with thermolysin and α-chymotrypsin. From these experiments a close resemblance to the locust AKH, a blocked decapeptide, is suggested.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.     

The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruchinae)

The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and eggs. Western blot analysis demonstrated that the majority of absorbed vicilins were degraded in the fat body. Tracing the fate of vicilins using FITC revealed that the FITC-vicilin complex was present inside cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus. Labelled vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These peptides are retained throughout the development of the insects and eventually are sequestered by the eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these peptides are known to have antimicrobial activity. Quantifications performed on internal organs from larvae of C. maculatus exposed to extremely dry seeds demonstrated that the vicilin concentration in the haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These results suggest that absorbed vicilins may also be involved in the survival of larvae in dry environments.     

The rôle of the suboesophageal ganglion in regulation of RNA synthesis in some insect tissues

The rôle of a factor produced by neurosecretory cells of the suboesophageal ganglion in the regulation of the level of RNA synthesis in some tissues of the female house cricket was studied in vivo and in vitro.It was shown that the action of the suboesophageal ganglion in vivo causes a reduction in the level of RNA synthesis in the neurosecretory cells of the pars intercerebralis, and also in the cells of the follicular epithelium, but it does not affect synthesis in the fat body cells.In experiments in which the action of the ganglion was examined in vitro, it was not found to have any influence on synthesis in the follicular epithelial cells, but the synthesis in the fat body cells was reduced. The causes of the differences obtained in the two types of experiments are discussed, and it is suggested that the suboesophageal ganglion acts directly on the fat body, but indirectly on the follicular epithelial cells through the corpora allata.     

The role of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda) in immune responses: A first survey

For the first time, a functional study of haemocytes from the crab Carcinus aestuarii was performed in order to evaluate their involvement in immune responses. Total haemocyte count (THC), phagocytosis, haemolymph opsonisation properties, hydrolytic and oxidative enzyme activities, and production of intracellular superoxide anion were evaluated. A great variability in THC was recorded among individuals, and haemocyte mean number was 6.4 (×10⁶) cells/ml haemolymph. Although only hyalinocytes were able to phagocytose yeast cells or Zymosan, phagocytic index was low (3%) and did not increase significantly (4%) after pre-incubation of yeast and Zymosan in cell-free haemolymph, suggesting that haemolymph did not have opsonising properties. All haemocyte types produced superoxide anion, whereas only granulocytes were positive to the hydrolytic enzymes assayed. In addition, only granulocytes were positive to phenoloxidase activity. Both Petri dish and spectrophotometric assays revealed a very low lysozyme-like activity in cell-free haemolymph (CFH) and haemocyte lysate (HL), although enzyme activity was higher in CFH than in HL. Interestingly, normalisation of data as to total protein content in CFH and HL resulted in an opposite situation, lysozyme-like activity being higher in HL than in CFH. This demonstrated that haemolymph of C. aestuarii has a high quantity of total proteins, functional properties of which need to be better investigated in future studies. Overall, the results obtained in the present study indicated that C. aestuarii haemocytes are not very active phagocytic cells, but they are more active in terms of both hydrolytic and oxidative enzyme activities and superoxide anion production.     

Water relations and haemolymph composition of two intertidal spiders (order araneae)

Water relations and haemolymph composition have been compared in two intertidal spiders which inhabit different zones of a rocky shore in the Cape Peninsula. Evaporative water losses of the mid-shore species, Desis formidabilis (O.P. Cambridge), are much greater than those of Amaurobioides africanus Hewitt, which occurs higher on the shore. Both species avoid desiccating conditions by remaining in silk-lined nests in suitable microclimates, and the nests of Desis formidabilis are submerged for a substantial portion of each tidal cycle. Osmolarities and sodium, potassium, magnesium, and chloride concentrations were measured for the haemolymph of both spiders. The haemolymph chemistry of Amaurobioides africanus resembles that of terrestrial spiders. The haemolymph of Desis formidabilis, however, has an osmolarity of 930 mOsm/l, a sodium concentration of 451 mM/l and a chloride concentration of 466 mM/l. It is suggested that this unusually concentrated haemolymph may represent an adaptation to a diet of marine crustaceans.     

Lipid composition of the fat body and haemolymph and its relation to lipid release in Oncopeltus fasciatus

Lipid composition of the fat body and haemolymph of male milkweed bug, Oncopeltus fasciatus, was determined. Triglycerides were the predominant lipids of the fat body while diglycerides accounted for the major lipid in the haemolymph. Sterols, sterol esters, and non-esterified fatty acids were present in both fat body and haemolymph besides triglycerides and diglycerides. Only traces of monoglycerides were detected.Gas chromatographic analysis of the fatty acids revealed a difference in the fatty acid composition between fat body and haemolymph glycerides and sterol esters. Oleate and linoleate were the predominant unsaturated fatty acids in both fat body and haemolymph lipids and in the milkweed seeds as well.When fat body was labelled in vivo and in vitro with ¹⁴C-palmitate, the fatty acid was incorporated largely into the triglycerides. When the prelabelled fat body was incubated with a medium containing haemolymph the fat body released lipids mainly as diglycerides. Some radioactivity was observed in the triglycerides and non-esterified fatty acids also.Electrophoretic analysis of the incubation medium containing the haemolymph revealed that the released lipids were bound to three haemolymph lipoprotein bands. Lipid mobilization, release, and transport in Oncopeltus are discussed in relation to studies on other insects.     

Effect of omeprazole-evoked hypergastrinemia on ultrastructure of enterochromaffin-like cells in the stomach of portacaval-shunted rats

Immunocytochemical staining of the nervous system of larva, pupa, and adult stage of Tenebrio molitor with anti-insulin serum demonstrated insulin-like peptides in the protocerebrum, corpora allata, and suboesophageal ganglion. During pupal development, marked changes in staining intensity of the protocerebral cells were detected. The staining pattern suggests release of insulin-like peptides early on day 0 and again on day 3 of the stadium. Injections of anti-insulin at these times caused significant delays in the timing of pupal/adult ecdysis. An immunoblot of haemolymph from day-3 pupae revealed a 6.5-kDa insulin-like molecule. These results suggest that the prothoracicotropic hormone of T. molitor is an insulin-like molecule.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

Control of juvenile hormone esterase activity in Galleria mellonella larvae

The juvenile hormone esterase (JHE) activity in Galleria mellonella larvae was measured after exposure to different experimental conditions that affect larval-pupal transformation. The data show that stimulation of production of JHE is closely coupled with the developmental signals that intiate larval-pupal metamorphosis. Injury, which delays pupation, delays the appearance of JHE activity if the larvae are injured within 48 hr after the last larval moult. Chilling of day-0 larvae induces a supernumerary larval moult and inhibits the appearance of JHE. However, JHE activity increases in chilled larvae when their commitment for an extra larval moult is reversed by starvation. Starvation is effective in reversing the commitment for an extra larval moult if commenced within 48 hr after chilling, thereby suggesting a critical period for that commitment. These data suggest that the stimulus for JHE synthesis and/or release occurs approximately within 48 hr after the last larval ecdysis. A series of studies involving implantation of brain, suboesophageal ganglion and fat body into chilled, as well as chilled and ligated larvae suggest that a factor from the brain is involved in stimulation or production of JHE in Galleria larvae.JH, which suppresses JHE activity in day-3, -5 and early day-6 Galleria larvae, stimulates the production of JHE in late day-6 larvae, suggesting that reprogramming in larval fat body may occur on day 6 of the last larval stadium.     

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N-acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.     

Haemolytic activity in the brown shrimp (Penaeus californiensis holmes) haemolymph

• 1.1. Natural haemolytic activity in brown shrimp (Penaeus californiensis) haemolymph was detected using mouse erythrocytes as target cells. This activity is unrelated to agglutinating and phenoloxidase activity, but it is another probable component of the shrimp defence system.
• 2.2. The haemolytic reaction is time and dose dependent, and a serine-protease is involved.
• 3.3. The haemolytic factor is thermolabile and has an apparent molecular weight of 23.5 kDa.

     

Evidence for site of biosynthesis and transport of the cyanoglucosides linamarin and lotaustralin in larvae of Zygaena trifolii (Insecta: Lepidoptera)

Zygaena larvae store relatively large amounts of cyanoglucosides within the haemolymph and cuticular cavities. Feeding experiments with ¹⁴C-labelled precursors demonstrate that valine and isoleucine are incorporated at different rates into the cyanoglucosides of the haemolymph and the defensive secretions. In conclusion from these different kinetics, we suggest that biosynthesis of cyanoglucosides takes place within the larval organs, such as fat body, gut, and/or haemolymph and that the epidermis is mainly involved in transporting and accumulating the cyanoglucosides within the integument.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

Haemolymph juvenile hormone III esterase activity during adult life of female and male crickets,Gryllus bimaculatus de Geer (Ensifera,Gryllidae)

Using an in vitro method, juvenile hormone III degradation was studied in the plasma of adult female and male crickets, Gryllus bimaculatus. The primary route of juvenile hormone III metabolism in cricket haemolymph is ester hydrolysis to juvenile hormone III acid by juvenile hormone III esterase. Juvenile hormone III esterase activity in females' haemolymph is low just after imaginal moulting. A sharp peak of enzyme activity is observed on day 3 after emergence, and two subsequent peak values occur in older animals. Plasma juvenile hormone III esterase activity in freshly ecdysed males is also low, but increases rapidly thereafter. Another increase in enzyme activity is observed in older males. The fluctuations in juvenile hormone III esterase activity are discussed in correlation with changes in haemolymph volume, haemolymph protein content, haemolymph juvenile hormone III titer, and the rates of juvenile hormone III biosynthesis in vitro of the corpora allata.     

Evidence and partial characterization of an inducible antibacterial factor in the haemolymph of Rhodnius prolixus

Antibacterial activity induced in the haemolymph of adult Rhodnius prolixus was investigated. Little or no antibacterial activity appeared in the first hours after Streptococcus mutans inoculation, but the activity increased reaching a maximum 5–6 days later and then declined gradually. The bacteria were destroyed in the haemolymph when the level of the antibacterial activity attained maximal value. The antibacterial activity appears to be related to the defence mechanism of the insect against bacterial infection. The activity was semi-purified by one-step Bio-Gel P-10 Gel-filtration and the estimated apparent mol. wt was 7,000 Daltons. It was found that this activity was due to a protein of small molecular weight. The activity was heat-stable, dialyzable and inactivated by trypsin treatment. The addition of haemolymph, obtained after inoculation of S. mutans into insects, to label-DNA growing cells (1) caused a dramatic reduction in the viable cells, measured by colony-forming units, and (2) increased approx 4-fold the releasing of labelled-DNA into the supernatant of the bacterial culture media. The possibility of this activity being lysozyme was excluded. The possible mechanism of action of this antibacterial activity is discussed.     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。