Interference-free SERS tags for ultrasensitive quantitative detection of tyrosinase in human serum based on magnetic bead separation.

Tyrosinase (TYR) expression and activity determine the rate and yield of melanin production. Studies have shown that TYR is a potential biomarker for melanoma and highly sensitive detection of TYR benefits early diagnosis of melanoma-related diseases. In this study, we developed a method that combines surface-enhanced Raman scattering (SERS) and sandwich-type immunity for sensitive detection of TYR, in which 4-mercaptobenzonitrile (4 MB) embedded between the Au core and Au shell (Au4MB @ Au) core-shell structure was employed as a SERS probe for quantitative detection of TYR while the magnetic bead serves as a capture substrate. Our results demonstrated that under magnetic separation, the specific SERS signal obtained is highly correlated with TYR concentrations. Furthermore, the combination of magnetic beads and Au4MB @ Au core-shell structure significantly improved the sensitivity of the sensing platform, resulting in detection limits of 0.45 ng mL-1. More importantly, the detection and analysis of TYR concentration in human serum samples showed good accuracy and an excellent recovery rate. Accuracy of the system was investigated from % recovery of spiked TYR standard solutions and found to be in the range of 90-104%, which further verified the feasibility and reliability of our method applied in a complex environment. We anticipate this SERS-based immunoassay method to be applied to TYR detection in the clinical setting and to be extended to other promising related fields.

A label-free luminescent assay for tyrosinase activity monitoring and inhibitor screening with responsive lanthanide coordination polymer nanoparticles.

In this work, a label-free, selective, and sensitive luminescent sensing platform was established for tyrosinase (TYR) activity monitoring and its inhibitor screening using one kind of lanthanide coordination polymer nanoparticles AMP-Tb/Ag+. By taking advantage of the specific binding and redox properties of Ag+ incorporated into the AMP-Tb network and dopamine (DA) as the product of the model substrate tyramine, the enzymatic reaction and the signal change of the sensing platform was effectively linked. The cooperative effect of a weakened energy transfer from AMP to Tb3+ by altering the electronic structure of Ag+ and an efficient photoinduced election transfer (PET) process caused by dopaquinone facilitated the luminescence quenching of Tb3+. Thus, this luminescent sensing platform could be employed for quantitative evaluation of TYR activity. There was a good linear range for TYR activity from 0.08 to 0.20 U mL-1 with a low detection limit of 0.004 U mL-1. Furthermore, this assay was successfully applied to accurate determination of TYR activity in human serum samples and efficient screening of TYR inhibitors. Considering unique spectral characteristics of lanthanides along with operation simplicity and superior analytical performance, this sensing platform is very promising in clinical diagnosis and drugs screening for TYR-associated diseases.

Dietary Protein and Carbohydrates Affect Immune Function and Performance in a Specialist Herbivore Insect (Manduca sexta).

Nutrition structures ecology and evolution across all scales of biological organization. It is well known that nutrition can have direct effects on performance and fitness, but indirect effects on physiological systems that mediate biotic interactions have been studied less frequently. Here, we focus on the interaction between nutrition, performance, and the immune system in a specialist herbivorous insect, Manduca sexta. We used a conceptual framework in nutritional ecology (the geometric framework) to examine how changes in diet quality affect aspects of the immune system used for defense against parasitoids. We raised caterpillars throughout their entire larval development on five different experimental diets that varied in protein and carbohydrate content and measured five aspects of the immune system: encapsulation, phenoloxidase activity, prophenoloxidase activity, total hemolymph protein, and hemocyte density. Overall, different parts of the immune function varied in response to interactions between carbohydrates, protein, and intake, but protein reductions had the largest impacts-mostly detrimental. In addition, our data suggest that diet quality mediates the relationship between performance (growth and survival) and immune function, as well as trade-offs among different components of immune function. Our work is the first to examine the interplay between nutrition, performance, and immune function with the geometric framework in a specialist insect herbivore.

Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

Simple construction of ratiometric fluorescent probe for the detection of dopamine and tyrosinase by the naked eye.

In this work, a simple and novel ratiometric fluorescence method based on ROX-DNA-functionalized CdZnTeS quantum dots (QDs) was developed for the detection of dopamine (DA) and tyrosinase (TYR). A ratiometric fluorescent probe was constructed by binding phosphorothioate DNA to the metal ions of QDs, which is a modification-free and low-cost method. DA was easily oxidized to DA quinone under the catalysis of TYR by dissolved O2, which effectively quenched the fluorescence of the QDs. Strong linear correlations were achieved for TYR in the range of 10.0-100.0 ng mL-1 and for DA in the range of 10.0-1000.0 nM. The limit of detection was estimated to be as low as 1.05 ng mL-1 for TYR and 1.93 nM for DA. Moreover, various colors were displayed in the course of detection, which could be observed by the naked eye. Therefore, an on-site and sensitive fluorescence method for the visual detection of DA and TYR can be developed. In addition, the findings revealed the potential applicability of the ratiometric fluorescent probe for the detection of DA and TYR in human serum. This ratiometric fluorescence method is not only sensitive and selective but also rapid and convenient for the detection of the analytes without sophisticated instrumentation.

A fluorescent sensor for detecting dopamine and tyrosinase activity by dual-emission carbon dots and gold nanoparticles.

In this work, we report a fluorescence strategy for detecting dopamine (DA) and sensing tyrosinase (TYR) activity on the basis of the dual-emission carbon dots (DECDs), which contain two emitters: the blue emitters (BE, maximum emission at 385nm) and yellow emitters (YE, maximum emission at 530nm). Gold nanoparticles (AuNPs) can effectively quench the two emissions of DECDs. The addition of DA aggregates AuNPs effectively, leading to the fluorescence recovery of dual emitters gradually. This strategy exhibits a high selectivity toward DA and shows good linear ranges, such as 0.5-3µM for BE and 0.1-3µM for YE. Additionally, the proposed method is successfully applied to the determination of DA in real samples with satisfactory recoveries. Subsequently, this DECDs-AuNPs platform is further taken advantage to assess TYR activity by the aid of TYR's capability for oxidation of DA into dopaquinone, which will not induce the agglomeration of AuNPs, so the fluorescence quenching of DECDs is associated with TYR activity. Finally, the mechanism of the reaction is discussed in detail, and the results suggest that both amine and phenolic hydroxyl groups of DA bring the aggregation of AuNPs.

Functionalized carbon quantum dots with dopamine for tyrosinase activity analysis.

Tyrosinase (TYR) is a key enzyme in melanin biosynthesis and its activity is an important biomarker for dermatological disorders, such as vitiligo, melanoma and actinic damages. Sensitive assay for TYR activity is significant for basic and clinical research. In this work, a facile fluorescent assay for TYR activity based on dopamine functionalized carbon quantum dots (CQDs-Dopa) has been developed. Dopamine (Dopa) was covalently bond to CQDs through a simple one-pot hydrothermal method, and the prepared CQDs-Dopa exhibited a fluorescence emission at 499 nm under exciting wavelength at 310 nm with a quantum yield of approximately 2.1%. When TYR was mixed with CODs-Dopa, the dopamine moiety in CQDs-Dopa conjugate was oxidized to O-dopaquinone, and an intra-particle photo-induced electron transfer (PET) process consequently occurred between CQDs and O-dopaquinone to quench the fluorescence of CQDs-Dopa. TYR activity can be determined based on the fluorescence quenching degree of CQDs-Dopa. This assay covered two broad linear ranges: 44.4-711.1 U L-1 and 711.1-2925.4 U L-1, with detection limit of 17.7 U L-1. The proposed fluorescent assay was applied to TYR activity measurement in human serum samples. It showed promising potential for TYR activity assay in clinical applications.

Immune responses in the haemolymph and antimicrobial peptide expression in the abdomen of Apis mellifera challenged with Spiroplasma melliferum CH-1.

Spiroplasma melliferum generally parasitizes honeybees and is one of main pathogens causing 'bee creeping disease' in China. Spiroplasma melliferum can be spread through honeybee pollination, which causes severe economic losses to apiculture. The design of this study was based on previous studies that utilized an in vitro bioassay to investigate the effects of S. melliferum CH-1 infection. We identified invasive S. melliferum CH-1 within Apis mellifera using transmission electron microscopy and investigated the immune response of honeybees infected with S. melliferum CH-1 by assaying the cellular immune response of the haemocytes, the plasma level of phenoloxidase activity and the transcript levels of 5 antimicrobial peptides, including the Abaecin, Apidaecin, Defensin 1, Defensin 2, and Hymenoptaecin gene products. The percentage of granulocytes in the haemolymph of infected honeybees was significantly higher than those of the controls during the early phase of infection, but the percentage of plasmatocytes was significantly higher than those of the controls at the fifth day post-infection. The phenoloxidase activity of the infected honeybees reached a maximum at the second day, and then decreased continuously. Moreover, the transcript levels of the 5 evaluated antimicrobial peptide genes were significantly increased during the early phase of infection and all 5 antimicrobial peptides were significantly decreased during the middle phase of infection. During the late phase of infection, only Defensin 2 and Hymenoptaecin showed significantly increased transcription. These results suggest that the honeybee immune responses could be activated by S. melliferum CH-1 during the early phase of infection and that S. melliferum CH-1 is also capable of circumventing the host defensive mechanisms to complete its life cycle within the honeybee during the middle phase of infection.

In Situ Fluorogenic and Chromogenic Reactions for the Sensitive Dual-Readout Assay of Tyrosinase Activity.

As a well-known copper-containing oxidase, tyrosinase has been anticipated to serve as the biomarker of skin diseases. We describe here an exquisite label-free fluorescent and colorimetric dual-readout assay of its activity, inspired by the specific oxidation ability of monophenolamine substrates to catecholamines and a unique fluorogenic reaction between resorcinol and catecholamines. By employing commercially available tyramine as the model substrate (dopamine as the product), it is found that the tyrosinase-incubated tyramine solution exhibits obvious pale yellow with intense blue fluorescence in the presence of resorcinol and O2, where the absorbance and fluorescence intensity are directly related to the concentration of added tyrosinase (i.e., the amount of conversion of tyramine to dopamine). The overall process of sensing tyrosinase activity takes less than 100 min at ambient temperature and pressure conditions with exceedingly simple operation procedure, explicit response mechanism, and formation of fluorophore with high quantum yield from scratch. Furthermore, such a convenient, rapid, cost-effective, and highly sensitive dual-readout assay exhibits promising prospect for the tyrosinase activity in extensive bioassays and clinic research as well as in screening potential tyrosinase inhibitors.

A simple and novel colorimetric assay for tyrosinase and inhibitor screening using 3,3',5,5'-tetramethylbenzidine as a chromogenic probe.

A novel colorimetric method for the detection of tyrosinase (TYR) and its inhibitor by taking utilization of Ag+-3,3',5,5'-tetramethylbenzidine (TMB) detection system has been proposed. Ag+ could oxidize TMB to oxidized TMB (oxTMB) and induce a blue color solution corresponding to an absorption peak centered at 652nm. The addition of dopamine (DA) could cause the reduction of oxTMB which resulted in the fading of the blue color and a decrease of the absorbance at 652nm. However, in the presence of TYR, DA could be oxidized to dopaquinone, which inhibited the reduction of oxTMB by DA, resulting in a blue color recovery and an increase of the absorbance at 652nm. Based on this finding, we propose a method to quantitatively detect TYR activity with the help of UV-vis spectroscopy. The developed assay is highly sensitive with a low detection limit of 0.010U/mL. More importantly, this method is fairly simple and inexpensive without the use of complicated nanomaterials. In addition, it constructs a useful platform for TYR inhibitor screening.

Phenoloxidase activity in humoral plasma, hemocyanin and hemocyanin separated proteins of the giant freshwater prawn Macrobrachium rosenbergii.

Hemocyanin is a copper containing protein and its role in the immune function of phenoloxidase (PO) activity was investigated in the giant freshwater prawn Macrobrachium rosenbergii. Hemocyanin, sedimented by ultracentrifugation from the plasma appeared on polyacrylamide gel electrophoresis (PAGE 7%) on Coomassie Brilliant Blue and bathocuproine sulfonic acid stain as four copper containing proteins of molecular masses 50, 60, 114 and 325kDa. Accordingly, on diethylaminoethyl-cellulose anion exchange column hemocyanin separated into four proteins designated as MrHc1, MrHc2, MrHc3 and MrHc4 with electrophoretically (PAGE) determined molecular masses of 60, 114, 50 and 325kDa respectively. The reduction of proteins in sodium dodecyl sulphate (SDS)-PAGE revealed that MrHc1 and 3 were monomeric for 60and 50kDa respectively, MrHc2 dimeric of 56 and 58kDa subunits and MrHc4 appeared with three subunits of 74, 76 and 78kDa. The PO activity was determined in plasma, hemocyanin and the four separated hemocyanin proteins in vitro using L-3,4-dihydroxyphenylalanine (L-DOPA) at pH7.5, 25°C and appeared elicited by exogenous activators such as trypsin, SDS, cell wall components of bacteria and polysaccharide laminarin. This study clearly demonstrated hemocyanin as the major copper containing protein in the plasma of M. rosenbergii with potent PO activity.

Allatotropin: A pleiotropic neuropeptide that elicits mosquito immune responses.

Allatotropins (AT) are neuropeptides with pleotropic functions on a variety of insect tissues. They affect processes such as juvenile hormone biosynthesis, cardiac rhythm, oviduct and hindgut contractions, nutrient absorption and circadian cycle. The present work provides experimental evidence that AT elicits immune responses in two important mosquito disease vectors, Anopheles albimanus and Aedes aegypti. Hemocytes and an immune-competent mosquito cell line responded to AT by showing strong morphological changes and increasing bacterial phagocytic activity. Phenoloxidase activity in hemolymph was also increased in Ae. aegypti mosquitoes treated with AT but not in An. albimanus, suggesting differences in the AT-dependent immune activation in the two species. In addition, two important insect immune markers, nitric oxide levels and expression of antimicrobial peptide genes, were increased in An. albimanus guts after AT treatment. AT conjugated to quantum dot nanocrystals (QDots) specifically labeled hemocytes in vivo in both mosquito species, implying molecular interactions between AT and hemocytes. The results of our studies suggest a new role for AT in the modulation of the immune response in mosquitoes.

Two-Photon Semiconducting Polymer Dots with Dual-Emission for Ratiometric Fluorescent Sensing and Bioimaging of Tyrosinase Activity.

Semiconducting polymer dots (Pdots) with one-, two-photon excitation and dual-emission have been synthesized by coprecipitation of two conjugated polymers including poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) and have been further functionalized with l-tyrosine methyl ester (Tyr-OMe) via electrostatic assembly for ratiometric fluorescent sensing and bioimaging of tyrosinase activity. Tyrosinase-catalyzed oxidation of Tyr-OMe effectively modulate the dual-emission fluorescence of PFO/CN-PPV@Tyr-OMe Pdots from orange to blue through a selective photoinduced electron transfer (PET) process. A two-photon ratiometric sensor at almost zero-background interference and bioimaging of tyrosinase activity have been demonstrated, suggesting the potential biomedical applications of the prepared functionalized Pdots.

miR-196a-2 rs11614913 polymorphism is associated with vitiligo by affecting heterodimeric molecular complexes of Tyr and Tyrp1.

Tyrosinase and tyrosinase-related protein 1 (Tyr-Tyrp1) complex plays a critical role in the synthesis of melanin intermediates, which involves the production of reactive oxygen species (ROS) and contributes to the development of vitiligo. Based on our previous observation that rs11614913 single nucleotide polymorphism (SNP) in miR-196a-2 could affect the risk of vitiligo by influencing Tyrp1, we hypothesized that the same SNP could also regulate the level of Tyr in vitiligo. The aim of this study was to evaluate the potential association between rs11614913 SNP in miR-196a-2 and serum Tyr level in vitiligo and the regulatory role of miR-196a-2 in the expression of Tyr in melanocytes. The serum Tyr level was detected in 116 patients with vitiligo and 116 controls by ELISA plate assay. The expression level of Tyrp1 and Tyr in PIG1(normal melanocyte cell lines) cells was analyzed by western blotting. The ROS level and apoptosis rate in PIG1 cells transfected with si-Tyr or control siRNA were tested by flow cytometry. The results show that the individuals with TT+TC genotypes in miR-196a-2 and higher Tyr level in serum had an increased risk of vitiligo compared with those who had the CC genotype and lower Tyr level (P < 0.001). Furthermore, the rs11614913 C allele in miR-196a-2 enhanced its inhibitory regulation on the expression of Tyr, the down-regulation of which in melanocytes successfully reduced the intracellular ROS levels and the apoptosis rate. In conclusion, our findings suggest that miR-196a-2 polymorphisms can regulate the Tyr levels, which influences the susceptibility of vitiligo.

Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris.

Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host-parasite interactions.

Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects.

To improve thermoregulation in colder environments, insects are expected to darken their cuticles with melanin via the phenoloxidase cascade, a phenomenon predicted by the thermal melanin hypothesis. However, the phenoloxidase cascade also plays a significant role in insect immunity, leading to the additional hypothesis that the thermal environment indirectly shapes immune function via direct selection on cuticle color. Support for the latter hypothesis comes from the cricket Allonemobius socius, where cuticle darkness and immune-related phenoloxidase activity increase with latitude. However, thermal environments vary seasonally as well as geographically, suggesting that seasonal plasticity in immunity may also exist. Although seasonal fluctuations in vertebrate immune function are common (because of flux in breeding or resource abundance), seasonality in invertebrate immunity has not been widely explored. We addressed this possibility by rearing crickets in simulated summer and fall environments and assayed their cuticle color and immune function. Prior to estimating immunity, crickets were placed in a common environment to minimize metabolic rate differences. Individuals reared under fall-like conditions exhibited darker cuticles, greater phenoloxidase activity and greater resistance to the bacteria Serratia marcescens. These data support the hypothesis that changes in the thermal environment modify cuticle color, which indirectly shapes immune investment through pleiotropy. This hypothesis may represent a widespread mechanism governing immunity in numerous systems, considering that most insects operate in seasonally and geographically variable thermal environments.

Influence of acute salinity changes on biochemical, hematological and immune characteristics of Fenneropenaeus indicus during white spot syndrome virus challenge.

The present study reports the influence of salinity (5, 15, 25 and 35 g/L) on the biochemical and immune characteristics of Fenneropenaeus indicus challenged with 5. 5 × 10(4) copy number of white spot syndrome virus (WSSV). F. indicus that had been reared in 25 g/L, injected with WSSV and transferred to 5, 15, 25 (control) and 35 g/L were examined after 0-120 hrs for total hemocyte count (THC), phenoloxidase (PO) and respiratory burst (RB) activity and alkaline and acid phosphatase activities. It was concluded that F. indicus that had been transferred from 25 g/L to lower and higher salinity levels (5, 15 and 35 g/L) had poorer immune indices and decreased resistance against WSSV infection. After 120 hrs, the mortality rate in WSSV-injected F. indicus experimental groups (5 and 35 g/L) was significantly higher than for F. indicus exposed to 25 and 15 g/L salinities. During the experimental period (0-120 hrs), biochemical variables, namely total protein, carbohydrate, and lipid concentrations, were measured in hemolymph of both experimental and control groups. Acute salinity changes induced an increase in protein variations across the tested salinity ranges in shrimp. After 24 hrs, THC and PO activity decreased significantly whereas RB, alkaline phosphatase and acid phosphatase activities increased in shrimps kept at the lower salinities of 5, 15 and 35 g/L.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri.

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

Detection of natural and induced phenoloxidase activities in human serum.

Natural and induced phenoloxidase activities were detected in human serum using nine different phenolic substrates, namely, tyrosine, tyramine, L-DOPA, DL-DOPA, dopamine, catechol, hydroquinone, protocatechuic acid and pyrogallol. Phenoloxidase activity was induced anew in serum using exogenous elicitors, such as proteases or detergents. Among the proteases and detergents tested, pronase, SDS and Tween 20 were the best elicitors of phenoloxidase activities in serum, wherein, hydroquinone was the best phenolic substrate for both untreated as well as pronase treated serum and SDS or Tween 20 treated serum resulted in highest oxidation of dopamine or tyrosine, respectively. In the present study, all these oxidative reactions were inhibited by phenoloxidase inhibitors, namely, PTU and tropolone, thereby, confirming the role of phenoloxidase in human serum.

Age- and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae).

As a result of the increased potential for disease transmission, insects are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a significant effect on either the darkness of the cuticle or antibacterial activity. This is the first report of age-related responses of adult insect immunity to crowding.