Host plant derived feeding deterrence towards ants in the turnip sawfly Athalia rosae

Larvae of the sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Tenthredinidae) feed on several glucosinolate-containing plants and have been shown to sequester the main glucosinolates of different hosts, namely sinalbin (p-hydroxybenzylglucosinolate) from Sinapis alba L., sinigrin (allylglucosinolate) from Brassica nigra (L.) Koch, and glucobarbarin ((S)-2-hydroxy-2-phenylethylglucosinolate) from Barbarea stricta Andrz. (Brassicaceae). These plant metabolites are stored in the haemolymph, which is readily released when larvae are attacked by predators. In a dual-choice bioassay the bio-activity of sawfly haemolymph collected from larvae reared on different host plants (S. alba, B. nigra, and B. stricta) was tested against the ant Myrmica rubra L. (Hymenoptera: Formicidae). The haemolymph had a stronger deterrence effect when the corresponding sawfly larvae were reared on S. alba than when reared on B. nigra and B. stricta. Haemolymph of caterpillars of Pieris rapae L. (Lepidoptera: Pieridae) that had fed on S. alba was not deterrent to the ants. No sinalbin could be detected in their haemolymph. The glucosinolates sinalbin and sinigrin, offered in a concentration comparable to that in the sawfly haemolymph, were deterrent to the ants, but not as strongly as the corresponding haemolymph samples. This suggests, that glucosinolates are not the only compounds involved in the chemical defence of A. rosae. However, the presence of sequestered glucosinolates is already a sufficient defence towards predators such as ants, and their effectiveness is modulated by the host plant chemistry.     

Major haemolymph proteins inCeratitis capitata: Biosynthesis and secretion during development

Summary The accumulation of major haemolymph proteins (a group of proteins immunologically related to Calliphorin) their biosynthesis in vivo and in organ culture as well as their secretion, has been studied during the late larval stages and white pupae of the Mediterranean fruit flyCeratitis capitata. The accumulation of major haemolymph proteins in the haemolymph, shows a twenty fold increase from the 4-day old larvae to the white pupae stage, while in the fat body there is only a seven fold increase. It is evident from the in vivo and organ culture studies, that the major haemolymph proteins are synthesized during the late larval stage and their synthesis declines abruptly during the stage of white pupae. It seems also that each polypeptide has its own characteristic developmental kinetics of synthesis. The major haemolymph proteins are synthesized in the fat body and are very quickly secreted into the haemolymph.     

Margolisiella islandica sp. nov. (Apicomplexa: Eimeridae) infecting Iceland scallop Chlamys islandica (Müller, 1776) in Icelandic waters

Wild Iceland scallops Chlamys islandica from an Icelandic bay were examined for parasites. Queen scallops Aequipecten opercularis from the Faroe Islands and king scallops Pecten maximus and queen scallops from Scottish waters were also examined. Observations revealed heavy infections of eimeriorine parasites in 95–100% of C. islandica but not the other scallop species. All life stages in the apicomplexan reproduction phases, i.e. merogony, gametogony and sporogony, were present. Trophozoites and meronts were common within endothelial cells of the heart’s auricle and two generations of free merozoites were frequently seen in great numbers in the haemolymph. Gamonts at various developmental stages were also abundant, most frequently free in the haemolymph. Macrogamonts were much more numerous than microgamonts. Oocysts were exclusively in the haemolymph; live mature oocysts contained numerous (>500) densely packed pairs of sporozoites forming sporocysts.Analysis of the 18S ribosomal DNA revealed that the parasite from C. islandica is most similar (97.7% identity) to an unidentified apicomplexan isolated from the haemolymph of the giant clam, Tridacna crocea, from Japan. Phylogenetic analyses showed that the novel sequence consistently grouped with the Tridacna sequence which formed a robust sister clade to the rhytidocystid group.We propose the name Margolisiella islandica sp. nov., referring to both type host and type locality.     

Haemolymph sugar levels in a nectar-feeding ant: dependence on metabolic expenditure and carbohydrate deprivation

In nectar-feeding insects, sugars are an important source of fuel and energy storage. Here, we analyzed the haemolymph sugar levels in foragers of the ant Camponotus rufipes trained to collect nectar from an artificial feeder, and their dependence on the metabolic rate during feeding. The main sugar found was trehalose, followed by glucose and traces of fructose and sucrose. In foragers, trehalose level was independent of their activity and metabolic rate while feeding. Carbohydrate deprivation of the colony had a strong effect on the haemolymph sugar levels of workers, with a significant decrease in trehalose and glucose with increasing starvation. We also found a correlation between haemolymph sugar levels and behavioral states, with immobile workers having higher trehalose and fructose levels than active ones. It is suggested that under food deprivation, inside-nest workers initially stay completely immobile as a strategy to save energy, and only become active and start to search for food when the trehalose levels decrease even more. Based on a conservative estimation, well-fed ants could travel up to 500 m, or spend more than 20 h inactive at 25°C, using only the energy provided by the haemolymph trehalose, before reaching the levels found in starved nest-mates.     

Peptidergic modulation of cardiovascular dynamics in the Dungeness crab,Cancer magister

Decapod crustacean pericardial organs contain extensive neurohormonal reserves which can be released directly into the haemolymph to act as physiological modulators. The present paper concerns the in vivo effects of two pericardial peptides, proctolin and crustacean cardioactive peptide, on cardiovascular dynamics in the crab Cancer magister. Infusion of proctolin into the pericardial sinus caused a slight decrease in heart rate concurrent with a large increase in cardiac stroke volume. It decreased haemolymph flow anteriorly through the paired anterolateral arteries and increased flow posteriorly and ventrally through the posterior aorta and sternal artery, respectively. The threshold for responses occurred at circulating concentrations of 10^-9 mol·l^-1, and haemolymph flows remained elevated for up to 30 min after peptide infusion. The effects of crustacean cardioactive peptide were less dramatic. Heart rate was not affected but a significant increase in stroke volume was observed. Crustacean cardioactive peptide increased haemolymph flow through the anterolateral arteries and increased scaphognathite rate. The threshold for crustacean cardioactive peptide activity was higher than for proctolin (10^-7 mol·l^-1 and 10^-6 mol·l^-1) but the responses to crustacean cardioactive peptide were of longer duration. The effects of proctolin on regional haemeolymph distribution in Cancer magister closely resemble the cardiovascular responses of this species when exposed to hypoxic conditions. These peptides may be implicated as cardiovascular regulators during environmental perturbations.     

The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta

Three out of 4 isolates of the Deuteromycete Metarhizium anisopliae were pathogenic for larvae of the tobacco hornworm, Manduca sexta. The most virulent isolate (ME1) grew sparsely in the insect prior to death and caused paralysis of its host. The other 2 pathogenic isolates killed Manduca larvae more slowly, grew profusely in the haemolymph and did not induce symptoms of toxicosis. Toxicosis is apparently due to the production by the fungus of several cyclodepsipeptide toxins, destruxins (DTX). ME1 produced large quantities of DTX in vitro, while other isolates produced less. Destruxin A (DTX A) was recovered from the haemolymph of paralysed, diseased insects infected with ME1, but not with other isolates. It is suggested that DTX may have a pathogenic role, when the toxins are active in causing disease, or an aggressive role, when they facilitate the establishment of the pathogen.     

Effects of Leucine-enkephalin on Catalase Activity and Glutathione Level in Haemolymph of the Scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

The study was designed to determine whether leucine-enkephalin (L-ENK) and delta receptor were present on the haemocytes of the scallop Chlamys farreri, and investigate the effects of L-ENK on the activity of catalase (CAT) and glutathione (GSH) level in haemolymph of the scallop. Lots of haemocytes immunoreactive to anti-L-ENK and -delta receptor sera were observed. The CAT activity and GSH level were investigated after 1, 5, and 50 μg/ml of L-ENK were added into the haemolymph of the scallop C. farreri. The CAT activity in haemocyte lysate supernatant (HLS) and supernatant were enhanced with increasing concentration of L-ENK. It was also found that the resultant HLS and supernatant GSH content was induced by L-ENK. Both the resultant HLS and supernatant CAT activity and GSH content was highest when the concentration of L-ENK was 50 μg/ml. By binding with opioid neuropeptide receptors, the opioid neuropeptides can regulate the intracellular and extracellular CAT activity and GSH content. Overall, the data strongly suggests an involvement of opioid peptides in the regulation and improvement of the antioxidant defence systems of the scallop C. farreri.     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

Heat shock response in mulberry silkworm races with different thermotolerances

The thermal sensitivity and heat shock response of the different races of the mulberry silkwormBombyx mori have been analysed. The multivoltine race, strainsC. Nichi andPure Mysore showed better survival rates than the bivoltine race, strainNB4D2 exposed to 41°C and above. In general, the fifth instar larvae and the pupae exhibited maximum tolerance compared to the early larval instars, adult moths or the eggs. Exposure up to 39°C for 1 or 2 h was tolerated equally whereas temperatures above 43°C proved to be lethal for all. Treatment of larvae at 41°C for 1 h resulted in a variety of physiological alterations including increased heart beat rates, differential haemocyte counts, enlargement of granulocytes and the presence of additional protein species in the tissues and haemolymph. The appearance of a 93 kDa protein in the haemolymph, fat bodies and cuticle, following the heat shocking of larvaein vivo was a characteristic feature in all the three strains examined although the kinetics of their appearance itself was different. In haemolymph, the protein appeared immediately in response to heat shock inC. Nichi reaching the maximal levels in 2–4 h whereas its presence was noticeable only after 2–4 h recovery time inPure Mysore and bivoltine races. The fat body from bothC. Nichi andNB4D2 showed the presence of 93 kDa, 89 kDa and 70 kDa proteins on heat shock. The haemocytes, on the other hand, expressed only a 70 kDa protein consequent to heat shock. The 93 kDa protein in the haemolymph, therefore could have arisen from some other tissue, possibly the fat body. The 93 kDa protein was detected after heat shock in pupae and adult moths as well, although the presence of an additional (56 kDa) protein was also apparent in the adults. The presence of 46 kDa and 28 kDa bands in addition to the 93 kDa band in the cuticular proteins immediately following heat shock was clearly discernible. The 70 kDa band did not show much changes in the cuticular proteins on heat shock. In contrast to the changes in protein profiles seen in tissues and haemolymph following heat shockin vivo, the heat treatment of isolated fat body or haemolymphin vitro resulted in protein degradation.     

Calcium exchanges in Anodonta cygnea: barriers and driving gradients

Electrical potential differences between the haemolymph and the extrapallial fluid, and between the haemolymph and the mantle cavity fluid, and ionic concentrations of calcium in the haemolymph and in extrapallial fluid were measured in vivo in Anodonta cygnea. The electrochemical potential of ionic calcium in the haemolymph is clearly above the electrochemical potential of ionic calcium in the environment and is very nearly in equilibrium with that of the extrapallial fluid. Simultaneous measurements of carbon dioxide partial pressure and pH in the extrapallial fluid showed that in this compartment ionic calcium is clearly above saturation. It is proposed that calcium deposition is regulated through the secretion of the organic matrix and by controlling the pH and the carbon dioxide partial pressure of the extrapallial fluid. An estimation of the minimum positive balance of calcium required to sustain shell growth together with the electrophysiological characterization of the mantle cavity epithelium showed that this tissue is not the route of entry of calcium into the animal.Abbreviations BW body weight - DW dry weight - E_EPF-S chemical potential difference - EPF extrapallial fluid - G_tot total conductance - I_sc short-circuit current - K_sp solubility product - MCE mantle cavity epithelium - MCF mantle cavity fluid - OME outer mantle epithelium - PCO₂ partial pressure of carbon dioxide - PVC Poly(vinyl chloride) - S shell - SEM standard error of mean - V _ic intracellular electrical potential - V _oc open-circuit voltage     

Invasion of the pathogenic fungus Metarhizium anisopliae through the guts of germfree desert locusts,Schistocerca gregaria

Less than 1% of an ingested inoculum of the pathogenic fungus Metarhizium anisopliae was retained for long enough (ca. 24 h) in the gut of the desert locust, Schistocerca gregaria, for germination and penetration to have occurred. The residual inoculum did not initiate an infection in guts of fed conventional or axenic locusts. However, symptoms of mycosis (hyphal bodies in the haemolymph, fungal penetration of the hindgut intima and epithelium, tetanic paralysis) were consistently observed in axenic but not conventional locusts which were starved post-inoculation.It is concluded that the antifungal toxin produced by the gut bacteria defends the desert locust against gut invasion by Metarhizium anisopliae during periods of starvation when the physical defences, prominent in fed insects, are less apparent.     

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri,the vector of huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama is currently threatening the citrus industry by transmitting the causative agent Candidatus Liberibacter asiaticus (CLas) of huanglongbing. Multiplication of CLas in haemolymph of D. citri indicates that it contains the necessary nutrients for CLas. Although many studies examine D. citri, the haemolymph composition of this dangerous pest remains to be investigated. In the present study, the haemolymph of D. citri is collected using a nanolitre syringe after the removal of one of its forelegs. The haemolymph is either derivatized with methyl chloroformate (MCF) or trimethylsilyl (TMS) derivatizing reagent and analyzed with gas chromatography–mass spectrometry. Nineteen amino acids, two organic acids and seven fatty acids are detected in the haemolymph after MCF derivatization. More metabolites are detected after TMS derivatization. Sugars are the most abundant metabolites in the haemolymph. Glucose and fructose are the main monosaccharides. Trehalose and sucrose are the major disaccharides. Furthermore, three inositol isomers (myo‐inositol, scyllo‐inositol and chiro‐inositol) are detected in the haemolymph. Organic acids are found in low amounts, whereas phosphoric acid is found at a higher concentration. Twenty‐four nucleotides and sugar nucleotides, including ATP, ADP and AMP, are detected using high‐performance anion‐exchange chromatography. Adenine nucleotides are the most abundant nucleotides followed by uridine and guanosine. The adenylate energy charge for the haemolymph is 0.77. Our results show that many metabolites found in the citrus phloem sap are also found in the haemolymph of D. citri.     

Water relations and osmotic pressure in Biomphalaria pfeifferi (Krauss), Biomphalaria glabrata (Say) and Helisoma trivolvis (Say) (Gastropoda : Planorbidae) in response to cationic alterations of the medium

The percentage of water in the total living body weight is restricted within a narrow range for each species, even in snails cultured under differing controlled ionic concentrations in the medium. The water level in Helisoma trivolvis is consistently higher than in Biomphalaria pfeifferi and B. glabrata under the same culture conditions.The whole cationic fraction of the total osmoconcentration of the haemolymph is maintained at a constant hypertonic level, even though individual cation levels vary with the cationic composition of the surrounding medium.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Seasonal variations of pH, pCO2, pO2, HCO3- and Ca2+ in the haemolymph: implications on the calcification physiology in Anodonta cygnea

A study about the relationship between the physical–chemical parameters and the calcium carbonate balance between the haemolymph fluid and mantle calcareous structures was carried out in Anodonta cygnea. An intense peak of HCO₃ ⁻ and a highest pH in December–January months may be understood as a preparation period for creating alkaline conditions. An intense pH decrease from January to February in parallel with the HCO₃ ⁻ reduction seems to indicate the beginning process of carbonate precipitation. On the other hand, the following calcium and HCO₃ ⁻ increases in February–May associated with a continuous and gradual pH fall profile may infer two combined aspects: calcium and HCO₃ ⁻ absorption from external environment and a simultaneous intense calcium carbonate deposition in the haemolymph. So, the pCO₂ peak in this period reflects a subsequent result on equilibrium balance between HCO₃ ⁻ absorption and deposition. The only significant pO₂ increase in the next period, from February to June, is related with an energetic increase to support the metabolic activity favouring the posterior intense pCO₂ peaks. The extended time of CO₂ production in the haemolymph from May to November should induce an increased metabolic acidosis with subsequent intense formation of both HCO₃ ⁻ and Ca²⁺ ions in the same period. This seems to result from CaCO₃ deposits dissolution in the haemolymph, the most direct calcareous source. Additionally, the later increase of metabolic succinic acid during autumn may greatly potentiate this acidosis increasing the dissolution process. Consequently, the pH profile present two simultaneous alkaline peaks in July and October, probably due to a strong HCO₃ ⁻ release from the CaCO₃ dissolution. So, the present seasonal results indicate that in the freshwater bivalve A. cygnea, the low metabolism with higher pH from the early winter is the main cause which may favour a calcareous precipitation, while the high metabolism with lower pH from the early summer may function as an inductor of calcareous dissolution in the haemolymph.     

Translation of the mRNAs coding for the major hemolymph proteins of Ceratitis capitata in cell-free system: comparison of the translatable mRNA levels to the respective biosynthetic levels of the proteins in the fat body during development

Four major hemolymph polypeptides (ceratitins) with molecular weights between 8.1 X 10(4) and 8.7 X 10(4) daltons have been identified in the fat body of late Ceratitis capitata larvae. Total fat body RNA from late larvae was translated in reticulocyte lysate, and the predominant in vitro translation products were shown to be the ceratitin precursors. The biosynthesis of these proteins during postembryonic development was studied in both tissue culture and cell-free system. Comparison of the biosynthetic patterns obtained in the two systems suggests a linear relationship between messenger concentration and protein synthesis. Three of these polypeptides show a coordinate pattern of synthesis and are immunologically related. After pupation, all four ceratitins are reabsorbed by the fat body where they accumulate.     

Acid-base and electrolyte regulation,and haemolymph gas transport in crayfish,Astacus astacus,exposed to soft,acid water with and without aluminium

Summary Intermoult crayfish (Astacus astacus) were exposed to acid (pH 4), soft water ([Ca⁺⁺]=100 mol·l^–1) in the absence and presence of aluminium (25 mol·l^–1) for variable time periods (up to 21 days) in order to assess the consequences for acid-base and electrolyte balance and haemolymph gas transport. Haemolymph osmolality and concentration of major ions decreased drastically and to a similar extent in acid and acid-aluminium water. Muscle tissue ion concentrations were, however, regulated at an almost constant level. A severe metabolic acidosis was gradually developed, attaining a haemolymph metabolic acid load of 6–7 mequiv·l^–1 after 12–21 days. The acidosis was partially compensated by ventilatory means, with the postbranchial haemolymph PCO₂ decreasing earlier in acidaluminium-exposed than in acid-exposed specimens. Hyperventilation seemed to be a direct acid-base regulatory response, since the rise in postbranchial PCO₂ had only minimal influence on haemolymph O₂ transport. The Bohr effect of Astacus astacus haemocyanin was low (log P₅₀/GdpH=-0.24), and the mean P₅₀ only increased from 15 to 19 mmHg after 21 days of acid exposure. The decrease in O₂ affinity with decreasing pH was accompanied by a decrease in the cooperativity of O₂ binding. The haemolymph haemocyanin concentration was not affected by acid and acid-aluminium exposure, but decreased after 21 days due to starvation. Muscle tissue aluminium concentrations were unaffected, whereas gill tissue concentrations increased in acid-aluminium exposed crayfish, most likely due to accumulation of aluminium on the gill surface. Mortality was low, and an internal hypoxia and lactacidosis was not developed in either of the experimental groups. This suggests that the gas transfer qualities of the chitincovered gills of crayfish are much less sensitive to acid and acid-aluminium stress than the gills of teleost fish.Abbreviations Hc haemocyanin - SO₂ saturation of Hc with O₂ - P ₅₀ oxygen tension of haemolymph at 50% SO₂ - n ₅₀ Hills coelficient around 50% SO₂     

Haemolymph amino acid and sugar levels in locusts fed nutritionally unbalanced diets

Aspects of pre- and post-ingestive compensation were investigated in locusts (Locusta migratoria) fed nutritionally unbalanced artificial diets containing 7% protein and 21% digestible carbohydrate (7:21) or 21% protein and 7% digestible carbohydrate (21:7). Feeding behaviour and haemolymph levels of amino acids and sugars were measured in locusts fed ad libitum on these diets. Locusts fed the high-protein diet had chronically elevated haemolymph levels of 15 out of 19 amino acids measured compared to locusts fed the low protein diet. However, haemolymph levels of lysine, alanine, aspartic acid and glutamic acid did not differ between diets, suggesting some specific regulatory mechanism for these amino acids. Haemolymph glucose and trehalose reflected levels of carbohydrate in the diets, being high in insects fed diet 7:21 relative to those given diet 21:7. These data are discussed in relation to the physiological and behavioural bases of nutritional homeostasis.Abbreviations AA amino acid(s) - PRO protein - CHO carbohydrate - PBS phosphate-buffered saline - MW molecular weight     

Quantitative studies on the release of locust adipokinetic hormone

Fractionation of methanolic extracts of haemolymph on Sephadex LH-20 made possible the measurement of the titre of adipokinetic hormone in the haemolymph of locusts. Experimentally produced high concentrations of haemolymph carbohydrate caused a delay in the mobilization of lipid during flight, and very low titres of the hormone were present in the haemolymph of locusts injected with trehalose immediately before a 25 min flight. In these locusts flight speed was higher than saline-injected controls. Although delayed lipid mobilization during flight was also seen in locusts injected with sucrose, sucrose is not utilized for flight metabolism and flight speed was not increased by the injection. Tentative estimates of the release rate (c. 1000pg/20min flight) and half life (c. 20 min) of adipokinetic hormone during flight are made. The results described suggest that during flight the rate at which trehalose disappears from the haemolymph does not play a major role in the initiation of the release of adipokinetic hormone.