Haemolymph Protein Concentration Research Articles

The Effect of Temperature on the Physiological Condition and Immune-Capacity of European Lobsters (Homarus gammarus) During Long-Term Starvation

Decapod crustaceans, such as those from the Homarus genus, are key benthic representatives supporting very valuable fishing and aquaculture industries. Those commercially caught, such as the European lobster (H. gammarus) can be stored in live facilities for short (few days) to long periods (up to 6 months) before being traded. Conditions in captivity are not standardized, including holding temperatures or feeding regimes. Herein, the physiological condition during long-term starvation (24 weeks) in H. gammarus was assessed at three temperatures (4, 8 and 12°C). Our results indicate that, H. gammarus have the capacity to endure long-term starvation. Principal component analysis (PCA) of measured parameters showed two main components (Eigen value>1). Fasted animals kept at 12°C separated from all others experimental groups due to higher total phenoloxidase (PO) activity in the hemolymph and water content in the muscle, suggesting that keeping H. gammarus un-fed at this higher temperature is physiologically more demanding and detrimental. This was later confirmed by significant changes particularly in this group in histology and lipid class composition of the hepatopancreas. These data call into question the suitability of current accepted in-vivo condition markers (e.g. hemolymph protein concentration) for determining the physiological condition and welfare of decapods such as H. gammarus.

English

Abstract The majority of Louisiana’s wild crayfish landings are harvested from the Atchafalaya River Basin (ARB) during floodplain inundation from the annual flood pulse. Spatial and temporal heterogeneity in ARB physicochemical characteristics are associated with flood pulse characteristics and floodplain inundation, and extensive areas of the ARB experience environmental hypoxia (dissolved oxygen [DO] < 2.0 mg·L-1) for several weeks to months during the annual flood pulse. The purpose of this research was to evaluate the effects of flood pulse characteristics and physicochemistry on harvested crayfish populations at 14 sites in the ARB that were sampled biweekly during the 2016 and 2017 crayfish seasons. Despite dissimilar 2016 and 2017 flood pulse characteristics, red swamp crawfish Procambarus clarkii and southern white river crawfish P. zonangulus carapace length and CPUE were similar between sample years. Comparisons of P. clarkii populations among physicochemical location groupings indicated that DO concentration, particularly chronically hypoxic water, is the principal abiotic variable influencing P. clarkii population characteristics. Although not significant, normoxic locations produced larger crayfish and yielded higher CPUE values for the majority of both crayfish seasons. Furthermore, hemolymph protein concentrations in P. clarkii from normoxic areas were significantly and consistently higher than individuals from chronically hypoxic locations.

Comparative Haemolymph Protein Content in Gen-3 Bivoltine Race of Bombyx Mori L. with Supplementation of Vitamin-C , E

Comparative Haemolymph Protein Content in Gen-3 Bivoltine Race of Bombyx Mori L. with Supplementation of Vitamin-C , E

Physiological Effect of Eyestalk Ablation on Nutrient Utilization and Plasma Protein Expression in the Female Giant Freshwater Prawn (Macrobrachium rosenbergii) during Different Molting Cycles

Eyestalk ablation is a common technique to induce spawning in marine shrimps. Currently, little is known about how this technique affects nutritional preferences (energy sources) in response to stress induced by eyestalk ablation during reproduction and growth. Thus, this study investigated the physiological effects of eyestalk ablation on hemolymph nutrient (glucose and protein) utilization and plasma protein expression of the female giant freshwater prawn (Macrobrachium rosenbergii). Sixty female M. rosenbergii prawns weighing 15.6 ± 0.3 g were divided into unablated (no eyestalk ablation, n = 30) and ablated (unilateral eyestalk ablation, n = 30) groups. The groups were then allowed to undergo reproductive and nonreproductive molting cycles. During normal molting, hemolymph protein (1,339.90 ± 95.08 mg/mL) concentrations decreased in the postmolt stage during the reproductive cycle, indicating that protein was used to attain full ovarian maturation, as evidenced by a large rise in the Gonadosomatic index value (8.47% ± 0.52%). The ablated group was vulnerable to stress, especially during the premolt (reproductive) stage, as indicated by a significant (P < 0.05) reduction in hemolymph protein concentrations, whereas no such stress-related effect was observed during the premolt (nonreproductive) stage. Interestingly, protein profiles were unaffected by eyestalk ablation, as shown by the similarity in profiles in both the unablated and ablated groups. Using a proteomic approach, fragment hemocyanin subunit 2, an immune-related protein, was identified in the ablated group. Overall, the results suggested that protein appeared to be the main nutritional source for reproduction and growth during short-term stress associated with eyestalk ablation, which led to stimulation of hemocyanin subunit 2.

Enzymes involved in immunity and characteristics of hemolymph in red sternum syndrome mud crabs (Scylla serrata)

The activities were compared of the immunological enzymes phenoloxidase (PO) and superoxide dismutase (SOD) and the concentration of proteins and urea nitrogen (UN) in the hemolymph of red sternum syndrome mud crabs, Scylla serrata, (Forskål, 1755) (Decapoda: Portunidae), with those of normal crabs. Twenty-five red sternum mud crabs and 10 normal mud crabs were randomly collected from crab farms in Samut Songkram province, Thailand. Statistical analysis used the mean ± SD and Student's t test at a significance level of α = 0.05. Levels of PO were 0.032 ± 0.013 units/mg protein and 0.075 ± 0.028 units/mg protein, while SOD levels were 0.493 ± 0.079 units/mg protein and 1.464 ± 0.783 units/mg protein, in normal and red sternum mud crabs, respectively. There was a significant difference in enzyme activities between the two groups. The hemolymph protein concentration in normal crabs was 1.03 ± 0.20 mg/mL which was significantly higher than in red sternum syndrome crabs (0.66 ± 0.23 mg/mL). The UN level in the hemolymph of normal mud crabs was significantly higher (10.78 ± 3.96 mg/dL) compared with that of red sternum mud crabs (4.59 ± 1.57 mg/dL). The findings revealed that the protein and UN levels in the hemolymph of red sternum mud crabs had decreased significantly. The red sternum syndrome was inversely related to the accumulated amount of protein and UN in crabs and had an extreme effect on protein metabolism.

Adipokinetic hormone and adenosine interfere with nematobacterial infection and locomotion in Drosophila melanogaster

This study examined how adipokinetic hormone (AKH) and adenosine affect defense responses in Drosophila melanogaster larvae infected with entomopathogenic nematodes (EPN, Steinernema carpocapsae and Heterorhabditis bacteriophora). Three loss-of-function mutant larvae were tested: Akh1, AdoR1 (adenosine receptor), and Akh1 AdoR1. Mortality decreased in all mutants post-EPN infection compared with the control (w1118). Additionally, co-application of external AKH with EPN significantly increased mortality beyond rates observed in EPN-only treatment, while also elevating carbon dioxide production, a measure of metabolism. Furthermore trehalose levels increased in both w1118 and Akh1 larvae post-EPN infection, but the latter group exhibited a lower increase and total trehalose levels. Interestingly, baseline trehalose was relatively high in untreated AdoR1 and Akh1 AdoR1 mutants, with levels remaining unaffected by infection. Infection also elevated haemolymph lipid content overall, but the different mutations did not substantially influence this change. In contrast, haemolymph protein content dropped after EPN infection in all tested groups, but this decline was more intense among Akh1. In uninfected larvae mutations decreased antioxidative capacity in Akh1 and increased in AdoR1, however, its post-infection increases were similar in all mutants, suggesting that antioxidant response in Drosophila involves mechanisms also beyond AKH and adenosine. Furthermore, AKH application in w1118 larvae significantly increased movement distance and percentage of larval activity, but reduced velocity. Mutations of Akh and AdoR did not strongly affect locomotion.

Toxic effects of Solanum xanthocarpum Sch &Wendle against Helicoverpa armigera (Hub.), Culex quinquefasciatus (Say.) and Eisenia fetida (Savigny, 1826).

Many commercially available agro and household chemicals are used as pesticides, repellents, and growth inhibitors against insect pests. The repeated uses of these chemicals against insect pests have caused the development of resistance in them; they also cause ill effects on nontarget organisms. The present study was aimed to evaluate the antifeedant, larvicidal, pupicidal, and biochemical effects of the solvent extracts of Solanum xanthocarpum against third instar larvae of Helicoverpa armigera. Hexane, chloroform and ethyl acetate extracts were subjected to phytochemical analysis. The results revealed the presence of terpenoids, flavonoid, and quinone. Maximum antifeedant activity of 72.30% was recorded in chloroform extract followed by hexane (69.02%) and ethyl acetate (57.40%) extracts against H. armigera. Chloroform extracts of S. xanthocarpum showed more than 60% larvicidal and pupicidal activity against H. armigera. The effective chloroform extract was fractionated with increasing polarity of solvent system (hexane, chloroform, and ethyl acetate extracts). Based on the TLC profile, nine major fractions were isolated. The fourth fraction showed higher antifeedant, larvicidal, and pupicidal activity against H. armigera. The effective fraction reduced the hemolymph and gut protein concentration in a concentration-dependent manner (r 2 0.99). The effective fraction 4 showed 100% larvicidal activity at 500ppm concentration with LC50 value of 227.95ppm. The fourth fraction did not show any toxic symptom or mortality of earthworm. Based on these results, this effective fraction could be used in the development of a pesticide formulation to control insect.

Hematological, physiological and genotoxicological effects of Match 5% EC insecticide on Biomphalaria alexandrina snails

Freshwater snails are used as brilliant biomarkers of aquatic ecosystem pollution by chemical compounds. The objective of this study is to highlight the ecotoxicological impacts of the insecticide Match 5%EC (its active ingredient is lufenuron 5% EC) on Biomphalaria alexandrina snails the intermediate host of Schistosoma mansoni in Egypt. The present investigation recorded a remarkable molluscicidal effect of lufenuron 5% EC on these snails and there was a decrease in total number of their hemocytes after exposure. Three morphologically distinct populations of circulating hemocytes were identified (round small cells, granulocytes and hyalinocytes) and results showed that some hyalinocytes had a shrunk nucleus and some were degenerated. Significant increase of transaminases (ALT and AST), while, a decrease of the total protein and albumin content in hemolymph was recorded. The results of alkaline comet assay in the present study demonstrated that lufenuron 5% EC has a genotoxic effect especially when its concentration increases. It can be concluded that Biomphalaria alexandrina snails can be used as bio monitor to screen the deleterious effects of lufenuron 5% EC insecticide as a cause of the environmental pollution, and this insecticide can be used in controlling schistosomiasis because of its molluscicidal effects on B. alexandrina snails.

Does sustained tolerance of reduced salinity seawater alter phagocytosis efficiency in haemocytes of the blue mussel Mytilus edulis (L.)?

Climate change driven increases in precipitation and extreme weather events bring with them periodic introductions of large volumes of freshwater to coastal waters and the possibility of extended salinity reductions capable of challenging marine organisms living there. Mytilus edulis is an osmoconformer, with no physiological capacity to maintain its haemolymph osmolarity above that of a dilute seawater medium. These mussels can however tolerate reduced seawater salinity to some degree by using intracellular volume control mechanisms to extend the range of seawater salinity they can tolerate, allowing them to continue to feed, respire and maintain general cellular function in fluctuating salinity conditions. When M. edulis are exposed to a falling salinity gradient, a critical salinity is reached that triggers closure of the valves, isolating their internal tissues from the surrounding environment and protecting them from further osmotic stress. This study investigated whether sustained tolerance of M. edulis to seawater with salinity just higher than the threshold level for valve closure affected haemocyte phagocytosis efficiency. Phagocytosis by haemocytes plays a significant role in the immune system of M. edulis by removing foreign bodies such as invading micro-organisms, together with dead and dying cells from the haemolymph. Valve gaping behaviour was recorded continuously in mussels exposed to a reducing seawater salinity gradient to establish the critical level initiating valve closure. M. edulis were then held for 5d within a flow through seawater system at a salinity of 25, close to the trigger value for valve closure in the population of mussels investigated (Namsenfjord, Norway). The exposed mussels continued to hold their valves open and active throughout, though mean valve gape and movement between valves were generally lower than control mussels held at ambient seawater with salinity of 34.3. Post-exposure total haemocyte counts and haemolymph protein concentrations in exposed mussels were similar to the controls, while phagocytosis performance, measured using flow cytometry, improved in the reduced salinity exposed mussels. These findings support a conclusion that M. edulis have the physiological capacity to continue to irrigate their gills for at least 5days when held continuously at seawater salinity close to their valve closure trigger level without detriment to haemocyte phagocytosis efficiency, a key component of their immune-response system. Such a capability will be important in the event of increasing persistent reduced salinity episodes.

Disturbing Effects of Three Insect Growth Regulators on General Body Metabolism of the Olive Leaf Moth, Palpita unionalis (Hübner) (Lepidoptera: Pyralidae)

The olive leaf moth Palpita unionalis (Lepidoptera: Pyralidae) is an economic pest of the commercial olive groves in Egypt and different Mediterranean countries. The present study was conducted aiming to assess the disturbing effects of three novel IGRs, viz. Novaluron, Methoxyfenozide and Pyriproxyfen (LC50 values: 0.97, 0.176 and 0.00009 ppm, respectively) on the main metabolites (proteins, carbohydrates and lipids) in haemolymph and fat bodies of larvae (24 h-, 48 h- and 72 h-post-treatment) as well as in the developed pupae (3-day, 6-day and 9-day old pupae). Both Novaluron and Pyriproxyfen prevalently enhanced the treated larvae to gain increasing protein content in haemolymph, but Methoxyfenozide enhanced only the 48 h-old larvae. In the fat bodies, all IGRs predominantly prohibited the treated larvae to attain normal protein content. All of the tested IGRs profoundly prevented the developed pupae to attain normal protein level, regardless of the age. The carbohydrate content in haemolymph of larvae was dramatically declined, regardless the age. In larval fat bodies, all of the tested IGRs exerted suppressing actions on the carbohydrate content. In the developed pupae, carbohydrate content had been decreased, regardless of the tested IGR or the pupal age. The total lipid content in the haemolymph of treated larvae was elaborately declined, regardless the larval age. In fat bodies, all IGRs induced the larvae to gain more lipids. In the developed pupae, the lipid content was pronouncedly increased by Novaluron and Methoxyfenozide but dramatically reduced by Pyriproxyfen.

Effect of Borrelial Infection on Haemolymph and Ovarian Protein Concentrations During Vitellogenesis in Ornithodoros erraticus, a vector of Relapsing Fever in Egypt

Abstract Changes in the total protein concentration of haemolymph and ovaries in Ornithodoros erraticus mated fed females uninfected and infected with Borrelia crocidurae were studied during vitellogenesis and at the end of oviposition. In the uninfected mated females, engorgement was followed by a decrease in the haemolymph total protein concentration immediately and for the following 2 days after feeding, then the level of haemolymph protein gradually increased on the following days to reach maximum levels on 5 – 9 d.a.f. The ovary total protein level increased gradually on the 1st d.a.f, then the increase continued evidently on the following days to reach a maximum on 9 d.a.f (day of oviposition).At the end of oviposition the level of haemolymph and ovaries total protein largely decreased. The increase in the level of haemolymph proteins was positively correlated with the increase in the ovary total protein, ovary weight and number of mature oocytes inside the ovary. Changes in the haemolymph and ovaries total protein concentration of mated females infected with Borrelia crocidurae followed the same pattern of the uninfected ones. However, borrelial infection reduced the level of haemolymph and ovaries total protein during most of the preoviposition period (3 – 9 d.a.f). Also the weight of ovary and number of mature oocytes were lower in the infected than uninfected on the 5th- 9th and 3rd-9thd.a.f, respectively. Freshly deposited eggs of the infected females had lower total protein concentration than those deposited by uninfected females.

Time Course Studies on Impact of Low Temperature Exposure on the Levels of Protein and Enzymes in Fifth Instar Larvae of Eri Silkworm, Philosamia ricini (Lepidoptera: satuniidae)

Lactate dehydrogenase (LDH; EC 1.1.1.27) and malate dehydrogenase (MDH; EC 1.1.1.37) are the enzymes involved in energy metabolism of Eri silkworm, Philosamia ricini. However, no previous study has been reported about effect of low temperature exposure on their levels in different tissues of Eri silkworm. The present study was aimed to the time-course effects of low temperature (~10°C) exposure of 5th instar P. ricini on the levels of protein and energy metabolism enzymes of three major tissues (haemolymph, silk gland and fat body). The Eri silkworm larvae, reared on fresh leaves of castor-oil-plant (Ricinus communis), were divided into 4 groups: a control group reared at 25 ± 2°C along with three experimental groups reared at 10 ± 1°C containing 50 larvae in each, for varying durations (2, 4 and 7 days). The cell free extract was prepared by centrifuging the tissue homogenate at 9000 g and used for biochemical estimations (total protein content, lactate dehydrogenase and malate dehydrogenase activities). For isozyme analysis, another set of homogenates (20% w/v) was prepared in buffer (0.2 M Tris HCl, pH 7.0) containing 0.2 M sucrose and 10 mM EDTA, and analyzed by native-PAGE followed by activity staining. The activities of lactate dehydrogenase and malate dehydrogenase showed significant decrease in haemolymph, whereas in fat bodies both enzymes showed increased activity. In silk gland, Lactate dehydrogenase activity decreased uniformly, whereas malate dehydrogenase activity increased at all exposure durations. The isozyme analysis revealed significant perturbations in their expression profiles. The low temperature exposure resulted into accumulation of protein content in haemolymph and depletion in silk gland and fat body tissues. Fat bodies emerged as the main energy producing organ under this condition. Lactate dehydrogenase and malate dehydrogenase displayed presence of only one isozyme in all the tissues tested. The isozyme behaviour of lactate dehydrogenase and malate dehydrogenase towards low temperature varied in different tissues. These results suggested that alterations in expression and functions of these enzymes might be associated to the acclimation of larvae at low temperature.

Metabolic effects of sinus and mandibular glands of the ghoust crab, Ocypode macrocera

The influence of sinus and mandibular glands on the metabolism of the Ocypode macrocerawas studied. Carbohydrate content of heart, hepatopancreas, muscles and hemolymph of eyestalk extract injected crabs was significantly higher than that of control, ablated and cheliped injected crabs (P&lt;0.05). Protein content of heart, gill, muscles and hemolymph of ablated crabs was also significantly higher than that of control, eyestalk extracted injected and cheliped injected crabs (P&lt;0.05). The result showed that the lipid content in the hepotopancreas (2.46 mg/g) and ovary (3.24 mg/g) of ablated animals was significantly higher when compared to control, eyestalk extract injected and cheliped injected crabs. Similarly carbohydrate content of gill, hepatopancreas and muscles of mandibular injected crabs was significantly higher than that of control and cheliped injected crabs (P&lt;0.05). Protein content of heart, gill, hepatopancreas, muscles and hemolymph of mandibular injected crabs was significantly higher than that of control and cheliped injected crabs. The result showed that mandibular extract injection decreased the lipid content of hepatopancreas and muscles.

Effects of Scleroderma sichuanensis Xiao (Hymenoptera: Bethylidae) venom and parasitism on nutritional content regulation in host Tenebrio molitor L. (Coleoptera: Tenebrionidae).

To explore the mechanisms by which the wasp Scleroderma sichuanensis Xiao regulates the physiology and biochemistry of its host, effects of S. sichuanensis venom and parasitism on host the Tenebrio molitor L. pupae were examined. Significant differences in nutritional content were noted between parasitized and non-parasitized pupae and between venom- and phosphate buffered saline-injected pupae. When pupae were injected with venom, the fat body could not be disintegrated into granules; however, when pupae were parasitized, fat-body disintegration occurred. Electrophoresis showed no differences in hemolymph protein content between parasitized pupae and those injected with venom, indicating that the wasp did not have narrow-spectrum peptides. These findings confirmed that S. sichuanensis was a typical idiobiont ectoparasitoid wasp, and that nutrient regulation was similar between idiobiont and koinobiont wasps. The strong similarities between the two treatments suggest that venom injection is a major factor responsible for changes in host nutrient content. The wasp fed mainly on reducing sugars, free amino acids, and fat-body tissues; larval fat bodies were derived from hemolymph and from host tissue. Our findings suggest that lipid catabolism might be accelerated, and that lipid biosynthesis might be inhibited, when host pupae are parasitized or injected with venom. In addition to venom, physiological and biochemical changes that occur during the parasitic process might be caused by venom, ovarian proteins, saliva, or secretions.

Alterations in protein and amino acid metabolism in honeybees (Apis mellifera) fed different l-leucine diets during the larval stage

l-Leucine is an important factor for growth and development in animals. However, very little is known about the nutritional role and physiological function of l-leucine in honeybees. In this study, we determined the effects of l-leucine supplementation on protein synthesis and amino acid balance in honeybee larvae. First instar larvae were fed different l-leucine diets (6.45, 8.45 and 10.45mg/g) during the entire larval stage. After the larvae egested urate crystals, we analyzed the protein and amino acid metabolism indexes in the larvae. There was little effect of l-leucine on larval body weight and body protein content, but the hemolymph protein concentrations were higher in 8.45 and 10.45mg/g groups than 6.45mg/g group. Compared to the 6.45mg/g group, l-leucine significantly increased the contents of Ala, Leu, Lys and Met in 8.45mg/g group and continued to be increased in 10.45mg/g group. l-Leucine affected many TOR signaling pathway- and l-leucine metabolism-related genes, including tor, 4Ebp1, bcaat et al. Moreover, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK) and lactate dehydrogenase (LDH) activities as well as the triglycerides (TG), total cholesterol (TCHO), uric acid (UA) and urea nitrogen (URER) contents in haemolymph were significantly different among treatments. These results suggested that many factors involved in the metabolism of l-leucine and larval hemolymph play important roles in honeybee protein synthesis and amino acid balance.

Conversion of protein from supplements into protein of hemolymph and fat bodies in worker honey bees (Apis mellifera L)

Adequate supplementation or replacement of pollen is necessary for maintaining healthy honey bee colonies during periods with limited pollen. Different supplemental protein diets could be metabolized in different ways by bees. We compared how protein supplements elaborated with free amino acids, with a soy-based diet, and with pollen, influence protein stores within individual honey bees. Caged bees were fed with one of three diets, and measurements were made based on protein content of hemolymph and fat bodies. The results show that these diets were metabolized in different way by bees. Bees fed with the free amino acid diet had significantly more proteins in hemolymph than bees fed with soya patty and pollen as they aged. The major amount of protein transported into the hemolymph by 27-day-old bees fed with free amino acid diet promoted the development of wax glands, and probably, it was the cause of a reduced storage of protein in fat bodies. Overall, our results suggest a connection between diets and physiological condition of honey bees.

Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming

Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to depend on the host-pathogen system, or the level of pathogen exposure and the type of dietary manipulation. Identifying whether immune priming or transgenerational effects are relevant in field populations, remains a challenge.

Climate change, nutrition and immunity: Effects of elevated CO2 and temperature on the immune function of an insect herbivore

Balanced nutrition is fundamental to health and immunity. For herbivorous insects, nutrient-compositional shifts in host plants due to elevated atmospheric CO2 concentrations and temperature may compromise this balance. Therefore, understanding their immune responses to such shifts is vital if we are to predict the outcomes of climate change for plant–herbivore–parasitoid and pathogen interactions. We tested the immune response of Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) feeding on Eucalyptus tereticornis Sm. seedlings exposed to elevated CO2 (640μmolmol−1; CE) and temperature (ambient plus 4°C; TE). Larvae were immune-challenged with a nylon monofilament in order to simulate parasitoid or pathogen attack without other effects of actual parasitism or pathology. The cellular (in vivo melanisation) and humoral (in vitro phenoloxidase PO activity) immune responses were assessed, and linked to changes in leaf chemistry. CE reduced foliar nitrogen (N) concentrations and increased C:N ratios and concentrations of total phenolics. The humoral response was reduced at CE. PO activity and haemolymph protein concentrations decreased at CE, while haemolymph protein concentrations were positively correlated with foliar N concentrations. However, the cellular response increased at CE and this was not correlated with any foliar traits. Immune parameters were not impacted by TE. Our study revealed that opposite cellular and humoral immune responses occurred as a result of plant-mediated effects at CE. In contrast, elevated temperatures within the tested range had minimal impact on immune responses. These complex interactions may alter the outcomes of parasitoid and pathogen attack in future climates.

Alternations in parasitological, biochemical and molecular parameters of Biomphalaria alexandrina snails, intermediate host of Schistosoma mansoni, induced post exposure to the proposed snail biocontrol agent Phasmarhabditis hermaphrodita nematode

ObjectiveTo elucidate the effect of the parasitic nematode Phasmarhabditis hermaphrodita (P. hermaphrodita) as snail biocontrol agent on biological, parasitological, biochemical and molecular parameters of Biomphalaria alexandrina (B. alexandrina) snails, the intermediate host of Schistosoma mansoni (S. mansoni). MethodsB. alexandrina snails (5–6 mm) were exposed to the 3rd stage larvae of the nematode P. hermaphrodita and/or S. mansoni miracidia for 24 h. Thereafter, they were thoroughly washed with clean dechlorinated water and transferred to clean aquaria for recovery and recording deteriorations in their biological, parasitological, biochemical and molecular parameters. ResultsThe results showed that snails were dead after 24 h of exposure to 100 nematode larvae/snail. Cercarial production/infected snail was reduced to 68 cercariae/snail post simultaneous exposure to 40 nematode larvae and 10 S. mansoni miracidia compared to control (783.7 cercariae/snail) (P < 0.001). Similarly, the total hemocytes of snails increased to 3 800 hemocytes/mL hemolymph by the 3rd day post exposure compared to 2 100 hemocytes/mL for control group (P < 0.001). The results, also, elicited a significant elevation in activities of the enzymes aspartate transaminase, alanine transaminase, acid phosphatase and alkaline phosphatase in hemolymph of snails exposed to nematode larvae. Similar pattern was recoded for activities of catalase, glutathione reduced and nitric oxide in snails' digestive gland. Meanwhile total protein and albumin concentrations in snails' hemolymph and lipid peroxide in digestive gland tissues were decreased. The electrophoretic analysis of ovotestis-digestive gland complex of snails exposed to the nematode larvae indicated an increase in RNA intensity, while no significant changes were recorded in the molecular weight of intact DNA compared to control snails. ConclusionsIt is concluded that exposure of B. alexandrina snails to P. hermaphrodita nematode deteriorates their biological, parasitological, biochemical and molecular parameters, rendering them to be unsuitable for schistosomiasis transmission. However, comprehensive tests are needed to determine efficacy of P. hermaphrodita as a schistosomiasis biocontrol agent aiming to incorporate in control program of this parasite.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels.

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.