Hemolymph Vitellogenin Levels Research Articles

Physiological responses to honeybee venom poisoning in a model organism, the firebug Pyrrhocoris apterus

In this study, the biochemical and physiological features of the firebug Pyrrhocoris apterus were investigated to understand the impact of the honeybee Apis mellifera venom on them using physiological methods (mortality, total level of metabolism), biochemical methods (ELISA, mass spectrometry, polyacrylamide gel electrophoresis, spectrophotometry) and molecular methods (real-time PCR). Together, the obtained findings suggest that venom injection increased the level of adipokinetic hormone (AKH) in the CNS of P. apterus, indicating that this hormone plays a key role in activating defence responses. Furthermore, histamine levels in the gut increased significantly after envenomation and did not seem to be modulated by AKH. In contrast, histamine levels in the haemolymph increased after treatment with AKH and AKH + venom. In addition, we found that vitellogenin levels in haemolymph decreased in both males and females after venom application. Lipids, which are the main energy metabolites used by Pyrrhocoris, were significantly exhausted from the haemolymph after the administration of venom and the co-application with AKH reversed this effect. However, we did not find much influence on the effect of digestive enzymes after the injection of venom. Our research has highlighted the noticeable effect of bee venom on P. apterus' body and provided new insights into the role of AKH in controlling defensive responses. However, it is also likely that there will be alternative defence mechanisms.

Effects of temperature and photoperiod on hemolymph vitellogenin levels during spawning events of the blue crab, Callinectes sapidus , in captivity

Effects of temperature and photoperiod on hemolymph vitellogenin levels during spawning events of the blue crab, <i>Callinectes sapidus</i> , in captivity

Alterations in the levels and distribution of octopamine in the central nervous system and ovary of the Pacific white shrimp, Litopenaeus vannamei, and its possible role in ovarian development

Octopamine (OA) is a major neurotransmitter that has not been studied in the Pacific white shrimp, Litopenaeus vannamei. Therefore, we investigated changes in OA levels, its distribution in regions of the central nervous system (CNS) and ovary during the ovarian maturation cycle, as well as its possible role in regulating ovarian maturation. OA exhibited the highest concentration in the brain and thoracic ganglia at ovarian stage II, and then declined to the lowest concentration at ovarian stages III and IV. In the cerebral ganglia, OA-immunoreactivity (OA-ir) was present in neurons of clusters 6, 17, the anterior and posterior medial protocerebral, olfactory, antenna II, and tegumentary neuropils. In the circumesophageal, subesophageal, thoracic ganglia and abdominal ganglia, OA-ir was detected in several neuropils, neurons and fibers. The high level of intensity in OA immunostaining was observed in early developmental stage of oocyte by comparison with low level of OA-ir in late stages of oocyte development. Functionally, OA-injected female shrimps at doses of 2.5×10−7 and 2.5×10−6mol/shrimp, showed significantly decreased gonado-somatic indices, oocyte diameters, and hemolymph vitellogenin levels, compared with control groups. This study showed changes of OA in the CNS and ovary reaching the highest level in early ovarian stages and declining in late stages, and it decreased hemolymph vitellogenin levels, suggesting significant involvement of OA in female reproduction in this species.

Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph.

Using vitellin monoclonal antibodies to assess the vitellogenesis-inhibiting hormone activity of Macrobrachium rosenbergii

Vitellogenesis in crustaceans is under the inhibitory regulation of the endocrine system in the X-organ/sinus gland complex in the eyestalks. In this study, two monoclonal antibodies specific to Macrobrachium rosenbergii vitellin and vitellogenin were generated to assess vitellogenesis-inhibiting hormone (VIH) activity. A combination of two monoclonal antibodies specific to the 89 and 100 kDa subunits of vitellin were used to develop a competitive enzyme-linked immunosorbent assay protocol for the quantitative determination of vitellogenin levels in haemolymph. Injection of eyestalk extracts from M. rosenbergii dissolved in either saline or in methanol-acetic acid caused a reduction in vitellogenin levels in the haemolymph at 12 and 24 h post-injection in bilateral eye-ablated prawns with developing ovaries. In contrast, the application of the heterologous eyestalk extract from Penaeus vannamei did not reduce vitellogenin levels in haemolymph. This assay is therefore specific and could be used to assess VIH activity during hormone purification.

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.

Effects of Bilateral and Unilateral Eyestalk Ablation on Vitellogenin Synthesis in Immature Female Kuruma Prawns, Marsupenaeus japonicus

In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.

Environmental physiology of the invasion of the Americas by Africanized honeybees

The expansion of Africanized honeybees (AHB) through the Americas has been one of the most spectacular and best-studied invasions by a biotype. African and European honeybees (EHB) hybridize, but with time, tropical and subtropical American environments have become dominated by AHB that exhibit only 20-35% genetic contribution from western European bees, and a predominance of African behavioral and physiological traits. EHB persist in temperate environments. Clines between AHB and EHB exist in ecotones of South and Central America, and are forming in North America. What individual-level genetic, behavioral and physiological traits determine the relative success of the AHB as an invader in the neotropics, and of the EHB in temperate areas? Preference for pollen versus nectar may be an important trait mediating these ecological trade-offs, as preference for pollen enhances nutrient intake and brood production for the AHB in the tropics, while a relative preference for nectar enhances honey stores and winter survival for EHB. AHB exhibit morphological (higher thorax-to-body mass ratios) and physiological (higher thorax-specific metabolic rates) traits that may improve flight capacity, dispersal, mating success and foraging intake. Enhanced winter longevity, linked with higher hemolymph vitellogenin levels, may be a key factor improving winter survival of EHB. Data from South America and distributions of AHB in the southwestern United States suggest that AHB-EHB hybrids will extend 200 km north of regions with a January maximal temperatures of 15-16°C. The formation of biotypic clines between AHB and EHB represents a unique opportunity to examine mechanisms responsible for the range limit of invaders.

Changes in gonadal development, androgenic gland cell structure, and hemolymph vitellogenin levels during male phase and sex change in laboratory-maintained protandric shrimp, Pandalus hypsinotus (Crustacea: Caridea: Pandalidae)

Most pandalid shrimps show protandric hermaphroditism, and male sexual differentiation is considered to be controlled by the androgenic gland. In the present study, we examined the histology of gonadal development during the male phase and sex change and the involvement of the androgenic gland in regulating male reproduction in laboratory-maintained Pandalus hypsinotus. Juvenile shrimps developed testicular tissues in the peripheral part of gonads during the age of 16–31 months and produced spermatozoa between 34 and 36 months. After reaching sexual maturity, male shrimps exhibited seasonal testicular development: active production of spermatozoa (February–May), disappearance of spermatozoa (spent, April–June), increase of spermatocytes (May–November), appearance of spermatids and spermatozoa in the gonads (November–February). The androgenic gland cells became larger and the rough endoplasmic reticulum in the cytoplasm developed at male sexual maturity. The cell structure shows that the androgenic gland hormone is a peptide. Furthermore, bilateral eyestalk ablation on immature male shrimps induced hypertrophy of the androgenic gland and acceleration of male sexual maturation. These results indicate the involvement of androgenic gland hormone and some eyestalk factor in regulating male sexual maturation. Over a 1-year laboratory-rearing period, some male shrimps (16.7%) changed sex. In transitional shrimps, testicular tissues in the gonads and androgenic glands degenerated; on the other hand, oocytes started yolk protein accumulation and hemolymph vitellogenin levels became high. These results suggest that androgenic gland degeneration is a trigger for sex change and that the vitellogenin level is a useful marker for sex change.

Hemolymph vitellogenin levels during final maturation and post-spawning in the female kuruma prawn, Marsupenaeus japonicus

A vitellogenin (VTG) ELISA was developed to clarify the relationship between hemolymph VTG levels and spawning conditions in female kuruma prawns. Hemolymph VTG was measured at the final maturation and spent stages. Collected hemolymph and oocytes were assessed for the 5 consecutive days of the spawning test. Of gravid females, 22.7% were broodstock, ready-to-spawn with cortical rods in oocytes. A negative correlation ( r 2=0.71) was found between the release rate of eggs and the hemolymph VTG concentration 1 day before spawning. Hemolymph VTG levels did not fluctuate significantly through the late granule, pre-maturation, and maturation stages. Late perinucleolus oocytes, which have already accumulated vitellin (Vn) in the cytoplasm, were found at the maturation stage. Positive immunoreactions against the anti-Vn serum were observed in the ovarian sections at the spent stage just after spawning. These results indicate that female prawns are preparing for the next spawning just before and after spawning, resulting in a high levels of hemolymph VTG throughout the late maturation and spent stages.

Ovarian Development and Hemolymph Vitellogenin Levels in Laboratory-maintained Protandric Shrimp, Pandalus hypsinotus: Measurement by a Newly Developed Time-resolved Fluoroimmunoassay (TR-FIA)

Most pandalid shrimps exhibit protandric hermaphroditism, and detailed information on ovarian development of pandalid species is important for a better understanding of vitellogenesis in crustacean species. In the present study, we characterized ovarian development under light and electron microscopy and examined the hemolymph vitellogenin levels in the coonstriped shrimp, Pandalus hypsinotus under laboratory conditions. To measure vitellogenin levels, a time-resolved fluoroimmunoassay (TR-FIA) was developed after purification of vitellin and production of the anti-vitellin antiserum. The TR-FIA showed wide assay range (0.98-2000 ng/ml), high sensitivity (0.5 ng/ml), and low assay variability (0.9-6.4% of intraassay coefficients, 1.4-5.1% for interassay coefficients). Female P. hypsinotus had non-vitellogenic ovaries in March after the eggs attached to the abdomen hatched, and started yolk accumulation in the ovaries during April-October. During yolk accumulation, yolk globules appeared and increased in the ooplasm. After yolk accumulation, gonadosomatic index (GSI) reached 8.3-8.5 just before oviposition. Females spawned and were ovigerous during June-July of the next year. Hemolymph vitellogenin levels were low (0.006+/-0.008 mg/ml, mean+/-SD) before the yolk accumulation, and became significantly higher (2.66 +/-0.93 mg/ml) during yolk accumulation (GSI, 2-8). Just before oviposition, levels declined to low levels (0.040+/-0.012 mg/ml). Vitellogenin levels were significantly correlated to GSI during the yolk accumulation. The obtained results show that the process of vitellogenesis during the female phase of P. hypsinotus is similar to other crustacean species that do not change sex.

Haemolymph metabolic variables in relation to eyestalk ablation and gonad development of Pacific white shrimp Litopenaeus vannamei Boone

The present study analyses some biochemical variables in haemolymph proposed as predictive indicators of the maturation capability following eyestalk ablation. Haemolymph of captive females was obtained before and 8 days after eyestalk ablation, and levels of haemocyanin, total proteins, glucose, lactate, cholesterol, triacylglycerides and vitellogenin were determined. Biochemical variables were also analysed in the hepatopancreas at the end of the experiment. Females were grouped as immature (previtellogenic stage) and mature (vitellogenic and cortical stages) based on histological analysis done 8 days after eyestalk ablation. To analyse haemolymph variables before eyestalk ablation in relation to maturation capability, immature females were classified as those with a low maturation capability and mature females as those with a high maturation capability. Females of high maturation capability had significantly higher vitellogenin levels before eyestalk ablation than females of low maturation capability. No significant differences were found for the other biochemical variables. Vitellogenin was also higher in mature than in immature females at the end of the experiment. These results indicate that vitellogenin levels in haemolymph could be used as possible predictive criteria of maturation capability, possibly because they reflect the degree of ovarian development at the time of eyestalk ablation.

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii.

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.

Development of an anti-vitellin ELISA for the assessment of reproduction in the ridgeback shrimp, Sicyonia ingentis

To investigate the reproductive regulation of the ridgeback shrimp, Sicyonia ingentis, vitellin (Vn) synthesis was studied. Using gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Vn was found to have a molecular mass of 322 kDa and to be composed of three subunits of 182, 91 and 85 kDa. Purified Vn was used to prepare anti-Vn antiserum, which was used to develop an enzyme-linked immunosorbent assay (ELISA) with a dynamic range of 0.3–300 ng. The ELISA was used to measure hemolymph levels of yolk proteins. The mean hemolymph concentrations in fresh caught animals ranged from 256 (±36.6 S.E.M.) to 1073 (±87.6 S.E.M.) mg/ml in stage 2 and 4 animals, respectively. The ELISA was also used to determine the effects of steroid hormone injections in adult non-reproductive female shrimp. One milligram injections of progesterone, 17α-hydroxyprogesterone or 17β-estradiol were administered for three consecutive days to individual females. There were no changes in hemolymph vitellogenin levels during the successive 7-day period following the first injection of any steroid.

Hemolymph vitellogenin levels and ovarian development during the reproductive and non-reproductive molt cycles in the giant freshwater prawn Macrobrachium rosenbergii

SUMMARY: In this study, ovarian development and vitellogenin levels were determined during the reproductive and non-reproductive molt cycles in the giant freshwater prawn Macrobrachium rosenbergii in order to examine the relationship between vitellogenesis and molting. During the reproductive molt cycle, the ovary developed synchronously with the advancement of molting stage. At molting stages B–C0, the ovaries contained oogonia and oocytes at the previtellogenic and endogenous vitellogenic stages, and hemolymph vitellogenin levels were relatively low (2.4–3.0 mg/mL). At stage C1, yolk accumulation commenced in oocytes at the endogenous vitellogenic stage, after which oocytes entered the exogenous vitellogenic stage. Vitellogenin levels increased (4.9 mg/mL) at this stage. During premolt stages D0–D3, vitellogenin titers were high (4.7–7.3 mg/mL), and gonadosomatic index (GSI) increased rapidly due to the yolk accumulation in the oocytes. Following ecdysis, yolk accumulation was completed at stage A0, and oviposition occurred. Entering stage A, vitellogenin levels became low (0.6–1.3 mg/mL). In contrast, throughout the non-reproductive molt cycle, GSI remained low, and oocytes did not develop beyond the previtellogenic or endogenous vitellogenic stage. Some atretic oocytes at the exogenous vitellogenic stage were observed in a few females. During the non-reproductive molt cycle, vitellogenin levels did not show distinct changes in relation to the molt cycle.

Hemolymph vitellogenin levels during ovarian development in the kuruma prawn Penaeus japonicus

SUMMARY: An enzyme immunoassay (EIA) system for kuruma prawn Penaeus japonicus vitellogenin (Vg) was developed and validated. In this assay, 96-well microtiter plates were coated with free vitellin (Vt). Assay conditions provided a detectable Vg range of 1.56–100 ng/well. The dose–response curve for serially diluted vitellogenic female hemolymph was parallel to the Vg standard curve. Male hemolymph produced absorbance values almost equal to background values, confirming the specificity of the assay. The intra- and interassay coefficient of variations were 6.0% and 10.5%, respectively. Using this assay, it was determined that very low levels of hemolymph Vg were detectable in prawns with oocytes at the previtellogenic stage. The Vg levels in the hemolymph rapidly increased at the endogenous vitellogenic stage and peaked at the early exogenous vitellogenic stage, but decreased at the late exogenous vitellogenic stage. Changes in hemolymph Vg levels preceded observed changes in gonadosomatic indices.