Natriuretic and depolarizing effects of a stable fly (Stomoxys calcitrans) factor on Malpighian tubules

A two-step HPLC purification procedure resulted in a factor from the stable fly that depolarizes the lumen-negative transepithelial voltage (V(t)) of the adult stable fly Malpighian tubule. When applied to tubules of the female mosquito, Aedes aegypti, this factor partially mimics the electrophysiological actions of the mosquito natriuretic factor (MNF). It also selectively increases active transepithelial Na transport by the mosquito Malpighian tubule. The blood meal causes a transient increase in hemolymph Na and Cl contents and hemolymph volume during the course of the 24-h post-feeding period. The level of a factor that is immunologically cross-reactive with the human atrial natriuretic peptide (ANP) increases more than 6-fold within 6h following a blood meal by the stable fly. The temporal pattern of the levels of the ANP-immunoreactive factor closely parallels the blood meal-induced rise and subsequent fall in hemolymph NaCl content and hemolymph volume, suggesting a functional correlation between the ANP-immunoreactive factor and the rate of NaCl and fluid loss from the hemolymph.     

Lipophorin levels in the yellow fever mosquito,Aedes aegypti,and the effect of feeding

High density lipophorin (HDLp) is the major lipid transport vehicle in insect hemolymph. Using an indirect ELISA, levels of HDLp were measured in the yellow fever mosquito, Aedes aegypti. The level of lipophorin, when normalized to the total weight of the insect, was similar in the different developmental stages. Starvation (access to water only) of adult females did not affect the level of HDLp nor its density when compared to sugar-fed females. On the other hand, blood feeding (of normally sugar-fed females) resulted in a three-fold increase of the HDLp level at 40 h after feeding. This increase was accompanied by a slight but significant increase in the density of HDLp at 24 h after feeding. Ingestion of a lipid-free protein meal or a lipid-supplemented protein meal induced changes in HDLp level and density that were comparable to those induced by ingestion of a blood meal. Ingestion of a blood meal, following starvation (access to water only) from the moment of adult emergence, did not induce an increase in HDLp level. The results presented indicate that, in contrast to other insect species, A. aegypti responds to an increased need for lipid transport in the hemolymph by increasing the amount of HDLp. Arch. Insect Biochem. Physiol. 34:301–312, 1997. © 1997 Wiley-Liss, Inc.     

Ionic and acid-base consequences of exposure to increased salinity in the zebra mussel, Dreissena polymorpha

Dreissena polymorpha, an invasive freshwater bivalve, displays physiological characteristics that reflect its ancestry in brackish water, yet it has limited ability to withstand modest increases in salinity. We examined changes in hemolymph ion concentrations and acid-base variables in mussels transferred to and incubated in 10% artificial seawater (ASW) for 7 days and then returned to pondwater (PW) for a further 7 days. Hemolymph was sampled (10 animals per sample period) every 4 h for the first 24-h incubation and at 72 h and 168 h for both the transfer to 10% ASW and the transfer back to PW. The initial response to transfer to 10% ASW was a rapid attainment of an apparent isoosmotic steady state, with most hemolymph ion concentrations rising and attaining steady state within 12 h. Hemolymph magnesium rose more slowly, and hemolymph calcium declined despite an increase in its concentration in the bathing medium. Hemolymph pH rose significantly during the first 24 h, from 7.96 to 8.25, as a result of increases in bicarbonate; pH subsequently returned to normal through increases in PCO2. When animals were returned to PW after 7 days' incubation in ASW, the response of the major hemolymph ions was largely the reverse of that effected by the transfer to ASW. Hemolymph pH was not altered significantly until after 72 h in PW, when declines in bicarbonate lowered the pH to 7.73. Strong ion difference (SID) was related significantly to hemolymph pH. Hemolymph calcium and magnesium showed a reciprocal relationship throughout both transfer and incubation. Solubility interactions between sulfate and calcium and magnesium may be important in determining calcium availability in solution. The Na/K ratio in hemolymph was maintained within relatively narrow bounds throughout the procedure and may contribute to the mussels' ability to volume-regulate during an osmotic challenge. Overall, the responses of D. polymorpha to modest changes in salinity were largely the result of passive processes.     

Movement of proteins across the digestive system of the tobacco budworm, Heliothis virescens

Bovine serum albumin (BSA) and anti‐BSA polyclonal antibody were used as model polypeptides to examine the movement of foreign proteins across the insect digestive system and their accumulation in hemolymph of fourth stadium tobacco budworms, Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Hydrateable meal pads were developed in these studies as a method for easily introducing compounds into the insect digestive system. When insects were allowed to feed continuously on hydrated meal pads containing 0.8 mg of anti‐BSA per gram diet, the level of antibody found in hemolymph was 2.4 ± 0.1 and 3.4 ± 0.1 µg ml^?1 (average  1 SEM) after 8 and 16 h, respectively, as determined by enzyme‐linked immunosorbant assay (ELISA). Continuous feeding on hydrated meal pads containing the same concentration of BSA produced hemolymph concentrations of 1.5 ± 0.1 and 1.6 ± 0.1 µg ml^?1 hemolymph at 8 and 16 h, respectively. Western blot analyses demonstrated that BSA and anti‐BSA both retained their primary and multimeric structure and that anti‐BSA maintained its antigenic activity in the meal pads and after movement from meal pads into the hemolymph. When 1 µg of anti‐BSA or BSA was injected into the hemocoel of fourth instars, the concentrations decreased with time and 120 min after injection were 20% and 0.6% of the original concentration, respectively. When added at the same concentration to plasma in vitro, the decrease was 81.5% and 57.5%, respectively, at 2 h. The accumulation of native anti‐BSA and BSA protein in insect hemolymph is the result of their rate of movement across the gut and their rate of turnover in hemolymph. Movement of anti‐BSA and BSA across the digestive system was also noted in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), Acheta domesticus (L.) (Orthoptera: Gryllidae), and Gromphadorhina portentosa (Schaum) (Blattaria: Blattellidae). Anti‐BSA and BSA were not detected in the hemolymph of Manduca sexta (L.) (Lepidoptera: Sphingidae) after feeding.     

The role of hemolymph proline as a nitrogen sink during blood meal digestion by the mosquito Aedes aegypti

Mosquitoes utilize the amino acids derived from blood meal protein to produce egg proteins. But the amino acids can also be used to produce egg lipid or can be oxidized for energy production. These latter two processes result in the release of nitrogen as toxic ammonia. Therefore, amino acids must be processed in such a way that amino acid nitrogen can be incorporated into non-toxic waste products. Proline is the predominant amino acid in the hemolymph of the adult female mosquito Aedes aegypti. After feeding on albumin meal, hemolymph proline levels increased five-fold over unfed levels, reached maximal levels in the first hours after feeding and remained high through oviposition. Hemolymph proline levels increased as the concentration of protein in the meal increased. When starved of sugar for 24 h prior to feeding on an albumin meal, hemolymph proline levels increased four-fold over the proline levels of non-starved mosquitoes. Proline levels after feeding on a protein deficient in essential amino acids, pike parvalbumin, increased to twice the levels of albumin fed mosquitoes. Based on these observations, we propose that mosquitoes utilize proline as a temporary nitrogen sink to store ammonia arising from deamination of blood meal amino acid.     

Expression of c-myc oncogene in rat liver by a dietary manipulation

After rats deprived of protein for several days are fed a meal containing protein, hepatic DNA replication is induced. When nuclear DNA synthesis is stimulated in the normally quiescent rat liver by a dietary manipulation, we examined the changes of the steady-state levels of messenger RNA for c-myc. Levels of c-myc mRNA are gradually elevated approximately 4 to 5-fold above normal in the livers of rats that are fed for several days a diet that lacks protein. After a nutritional shift from a protein-free diet to a diet containing 50% casein, the levels of c-myc mRNA decrease rapidly by 2 h and returned to approximately basal levels after 8 h. Our results suggest that c-myc expression during the prereplicative stage of liver is likely to reflect events associated with entry and progression of hepatocytes into the cell cycle.     

Functional morphology of adult female Culex quinquefasciatus midgut during blood digestion

The adult female Culex quinquefasciatus midgut comprises a narrow anterior and a dilated posterior region, with epithelia composed of a monolayer of adjacent epithelial cells joined at the apical portion by septate junctions. Densely packed apical microvilli and an intricate basal labyrinth characterise each cell pole. Our morphological studies suggest that, during blood digestion, the anterior midgut region also participates in an initial absorptive stage which is probably related to the intake of water, salts and other small molecules. This activity peaked by 6h after bloodmeal feeding (ABF) and ended approximately 18 h ABF, when the peritrophic membrane was already formed. After this time, absorption only occurred in the posterior region, with morphologic and biochemical evidence of high synthetic activity related to the secretion of proteases. Chymotrypsin, elastase, aminopeptidase, and trypsin reached their maximum activity at around 36 h ABF. Digestion products were apparently absorbed and transported to the basal labyrinth, from where they should be released to the hemolymph. At 72 h ABF, proteolysis had already ended and protein levels had returned to those observed before blood meal. The epithelium of the posterior region, however, did not return to its initial morphology, appearing quite disorganised. Additionally, from 48 h ABF onwards some epithelial cells showed morphological signals of apoptosis.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

Fate of GFP-expressing Escherichia coli in the midgut and response to ingestion in a tick, Ornithodoros moubata (Acari: Argasidae)

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.     

Lipid metabolism in the cockroach, Periplaneta americana, is activated by the hypertrehalosemic peptide, HTH-I

Phosphatidylcholine and phosphatidylethanolamine are the major constituents of the phospholipid pool in cockroach (Periplaneta americana) fat body and hemolymph. Both species of phospholipid are significantly decreased 6h after injecting hypertrehalosemic hormone I (HTH-I) into the hemocoel. Loss of phospholipid is accompanied by an accumulation of the phospholipid degradation products glycerophosphorylcholine and glycerol. HTH-I also increases phospholipase activity in the hemolymph and this is thought to be responsible for the depletion of hemolymph phospholipid. Phospholipase activity peaks approximately 2h after injection of HTH-I and returns to normal at 6h. In vitro, total phospholipid in the fat body is decreased by HTH-I whereas the concentration of diacylglycerol displays a corresponding increase. HTH-I elevates free fatty acid levels but has no effect on triacylglycerol. These effects of HTH-I are blocked by the phospholipase inhibitor mepacrine.     

Hemolymph of Anopheles stephensi from noninfected and Plasmodium berghei-infected mosquitoes. 3. Carbohydrates.

Determinations were made of carbohydrates in hemolymph collected from adult female mosquitoes (Anopheles stephensi). First the hemolymph was fractionated by extraction and precipitation procedures, after which qualitative and quantitative determinations of carbohydrates were made by thin layer chromatography. The most abundant sugars found in the hemolymph were glucose and trehalose, though maltose, glucuronic acid, and inositol could be found after the mosquitoes took blood meals. After the mosquitoes ingested a noninfected blood meal, their hemolymph sugar levels rose almost 4-fold. There was less of an increase following a blood meal infected with the rodent malaria parasite, Plasmodium berghei. Depletion of sugars in the hemolymph of infected mosquitoes may result from direct utilization of sugar by the malaria parasite developing within the mosquito.     

Neuropeptide F and its expression in the yellow fever mosquito,Aedes aegypti

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal.     

Juvenile hormone and juvenile hormone esterase in adult females of the mosquito Aedes aegypti

Juvenile hormone III levels and juvenile hormone esterase activity were measured in whole body extracts and haemolymph, respectively, of female Aedes aegypti. The amount of juvenile hormone, determined by coupled gas chromatography-mass spectrometry, rose over the first 2 days after emergence from 0.7 to 7.5 ng/g, and then slowly fell over the next 5 days in females not given a blood meal. In females fed blood, juvenile hormone levels fell during the first 3 h to 2.3 ng/g. The rate of decline then slowed so that levels had reached their lowest point (0.4 ng/g) by 24 h after the blood meal. By 48 h, levels started to rise again until 96 h when they were equivalent to pre-blood meal levels.Juvenile hormone esterase activity in the haemolymph of females was measured with a partition assay. The esterase activity showed small fluctuations in unfed animals. In females fed blood on the 3rd day after emergence, the juvenile hormone esterase activity rose slowly to a peak at 36 h. At 42 h it began to decline, and by 66 h it had returned to pre-blood meal levels. Thus, juvenile hormone levels and juvenile hormone esterase activity were inversely correlated after a blood meal. Both the ovary and fat body produce juvenile hormone esterase in organ culture.Juvenile hormone III acid was the only metabolite produced after incubation of haemolymph with racemic-labelled juvenile hormone III. Juvenile hormone acid, diol, and acid diol were the main metabolic products seen in whole animal extracts after topical application of labelled hormone. About 25% of topically applied, labelled juvenile hormone appears in the haemolymph as the acid diol, and 50% of this is excreted in the urine immediately after the blood meal. Topical application of BEPAT (S-benzyl-O-ethyl phosphoramidothiolate), a specific inhibitor of juvenile hormone esterase, resulted in the absence of juvenile hormone acid and a reduction in the acid diol. Both BEPAT and methoprene, a juvenile hormone analogue, caused a reduction in egg hatch when applied topically 30 h after a blood meal, demonstrating that the decline in juvenile hormone levels after a blood meal is necessary for normal egg development and suggesting that the decline is mediated, at least in part, by juvenile hormone esterase.     

Reversible alteration of morphology in an invertebrate erythrocyte: properties of the natural inducer and the cellular response

The normal shape of the erythrocytes of the bivalves known as blood clams is maintained by a marginal band (MB) of microtubules. When hemolymph (or "blood") is withdrawn from the animal, its erythrocytes change, within minutes, from the normal smooth-surfaced, flattened ellipsoids (N-cells) to spheroids with folded surfaces (X-cells). This alteration can be prevented by rapidly diluting the hemolymph with physiological medium, yielding N-cells for use in studying the transformation to X-cells. Bioassays showed that shape transformation was induced by a hemolymph activity (Hx) and was a function, in part, of cell responsiveness to this activity. Eventually the shape of the cells spontaneously returned to normal, at a rate dependent upon the concentration of the cells and of Hx; recovery was correlated with loss of Hx. The X-cells contained an intact but highly deformed MB, but this was not the effector of the transformation. Erythrocytes made to lack MBs still changed shape, although they did not recover as completely as did the MB-containing controls. When clams were cooled before hemolymph was withdrawn, the concentration of Hx was reduced. Hx was retained after dialysis of hemolymph, and initial filtration and chromatography indicated that its Mr was greater than 500,000. Shape transformation was blocked by EGTA, by serine protease inhibitors, and by sodium azide; the last indicates ATP-dependence. Although the mechanism responsible for shape transformation remains to be determined, the data suggest that the change is triggered by a coagulation-related activity in response to the removal of hemolymph from the animal.     

Hemolymph of Anopheles stephensi from uninfected and Plasmodium berghei-infected mosquitoes. 2. Free amino acids.

Determinations were made of free amino acids in hemolymph collected from adult female Anopheles stephensi mosquitoes. The hemolymph first was fractionated by extraction and precipitation procedures, after which qualitative determinations of free amino acids were made by high voltage thin layer electrophoresis, and thin layer chromatography. Subsequent quantitative determinations were made with an automatic amino acid analyzer. The concentration of total free amino acids in the hemolymph rose 60--70% after the mosquito took a blood meal, and remained relatively constant thereafter. When mosquitoes took a blood meal infected with the rodent malaria parasite Plasmodium berghei, the rise in total free amino acids was only 15--25%. The chief differences that occurred with individual free amino acids was that infected mosquitoes had greater increases in arginine, greater decreases in valine and histidine, and a total loss of detectable methionine.     

Ploidy levels and DNA synthesis in fat body cells of the adult mosquito,Aedes aegypti: The role of juvenile hormone

Adult females of the mosquito Aedes aegypti showed two cycles of DNA replication in the fat body based on microspectrophotometric measurement of changes in nuclear DNA. The first cycle began after emergence and resulted in 80% of diploid fat body cells becoming tetraploid and 20% becoming octoploid by the end of the third day. The second replication cycle occurred 48–72 h after a blood meal and resulted in an increase in octoploid nuclei to 67% Topical application of juvenile hormone or methoprene to abdomens isolated at emergence stimulated an increase in ploidy levels above that normally seen in situ. Synthesis of DNA, estimated by incorporation of injected [³H]-thymidine, rose after emergence and remained high for 2 days. Synthesis increased again after a blood meal, reached a peak by 6 h, and returned to low levels by 24 h after the meal. The timing of DNA synthesis and a measurable increase in ploidy were temporally separated. The ploidy increase, but not DNA synthesis, was correlated with increases in juvenile hormone levels.     

Water intake induced by water deprivation in the quail,Coturnix coturnix japonica

Mechanisms inducing drinking after water deprivation, and mechanisms terminating drinking after rehydration, were investigated in the quail, Coturnix coturnix japonica. 1. Water intake was induced after 4 h of water deprivation, and the amount of water drunk increased in proportion to the period of water deprivation. Drinking occurred immediately after deprivation. Drinking occurred immediately after deprived birds were given access to water, and continued for periods proportional to the period of water deprivation. 2. Plasma angiotensin II concentration increased, as did plasma osmolality and Na+ concentration, and blood volume decreased after water deprivation. The increase in plasma angiotensin II concentration and decrease in blood volume occurred soon after the start of water deprivation, whereas plasma osmolality and Na+ concentration did not increase until at least 4 h after the start of water deprivation. 3. These results indicate that extracellular dehydration and angiotensin II are responsible for the significant drinking that follows 4 h of water deprivation, and that cellular dehydration is also involved in the stimulation of drinking that occurs after longer periods of water deprivation. 4. Plasma osmolality and Na+ concentration in birds deprived of water for 48 h quickly returned to normal levels after the birds were allowed access to water. Plasma angiotensin II levels and blood volume also approached the values measured prior to water deprivation. However, the rate and degree of restoration of normal values were reduced, and normal values were not restored even after 1.5 h or rehydration when drinking terminated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amblyomma americanum: specific uptake of immunoglobulins into tick hemolymph during feeding

The passage of immunoglobulins in the blood meal into hemolymph prompts development of vaccines against internal antigens of ticks, but little is known about kinetics and specificity of the immunoglobulin uptake. We used capillary feeding of adult Amblyomma americanum hard ticks to introduce compounds into the midgut and then examined the hemolymph after various times for their presence and concentration. Immunoglobulins of different sources, albumin, choramphenicol acetyltransferase, inulin, and mannitol were labeled with (125)I, (14)C, or biotin. With the exception of the carbohydrate inulin, all the compounds entered the hemolymph of tick during capillary feeding. The small molecule mannitol had the highest rate of entry at 9% after 6 h. Among proteins, the entry of immunoglobulin G (IgG) of different species into the hemolymph was greater at 6% after 6 h than for the smaller proteins albumin or choramphenicol acetyltransferase at 1 and 3%, respectively. The entry of denatured IgG was equal to that of nondenatured protein. There was no evidence of degradation of the IgG or of its binding to cells once it entered the hemolymph. A monoclonal IgG antibody labeled with biotin entered the hemolymph and retained its ability to bind to its specific antigen in an immunoassay. Although different proteins entered the hemolymph after capillary feeding, there was evidence of a specific mechanism for immunoglobulin uptake.     

Effects of salinity manipulations on blood pressures in an osmoconforming chordate,the hagfish, <Emphasis Type="Italic">Eptatretus cirrhatus</Emphasis>

Arterial and venous pressures were measured in hagfishes subjected to acute changes in salinity. The osmotic pressure of the seawater (SW) was increased or decreased by approximately 10%. Sixty minutes after the change in medium osmolarity the osmotic pressure of the blood corresponded with that of the medium. Following transfer to 90% SW all measured parameters changed as predicted for a passive increase in blood volume, apart from the pressure in the posterior cardinal vein (PCV) which fell. By 2 h dorsal aortic (DA) pressure and pressure in the PCV and supraintestinal vein had returned to pre-change values. In contrast, following exposure to 110% SW, pressures fell and apart from the supraintestinal vein they remained low at 120 min. At 24 h, DA pressure was lower than pre-change values for both groups. The data are consistent with the concept of central venous tone being regulated in hagfishes, which cope better with volume expansion than volume depletion.     

Activity of the corpora allata of adult female Aedes aegypti: effects of mating and feeding

The synthesis of juvenile hormone III (JH III) by the isolated corpora allata (CA) of Aedes aegypti adult female was studied using an in vitro radiochemical assay. We dissected the corpora allata-corpora cardiaca (CA-CC) complex attached to a piece of aorta. The complex was left connected to the intact head capsule to facilitate the visualization and transfer of the glands. A linear increase in the cumulative amount of biosynthesized JH III was found for at least the first 6 h of incubation; approximately 45% of the synthesized JH III was present in the medium. There was a dependence of JH III synthesis on exogenous methionine supply. Using reversed phase high performance liquid chromatography two major labeled products biosynthesized by the CA were separated. They co-migrated with JH III and methyl farnesoate (MF). The identity of the biosynthesized JH III was confirmed by gas chromatography-mass spectrometry. JH III synthesis was only 2.0 fmol/pair gland/h immediately after adult emergence, but increased to 32.6 fmol/ pair gland/h 18 h later in sugar-fed females. Two days after emergence, the CA biosynthetic activity slowly started to decrease, and reached values of around 5.3 fmol/pair gland/h by one week after emergence. Synthesis of JH was similar from either sugar-fed females mated or unmated. A blood meal resulted in a decrease of JH III synthesis in CA from mated females by 12 h after feeding and from virgin females by 24 h after feeding. JH III biosynthesis remained low for at least 96 h in mated females, but was back to higher levels 72 h after feeding in virgin females. Rates of JH III biosynthesis closely reflected the hemolymph levels of JH III both after emergence and after a blood meal described by Shapiro et al. (1986). The activity of the CA in Aedes aegypti females seems to be regulated by developmental changes and nutritional signals, and to be independent of mating stimulus.