Hemocyte Concentration Research Articles

Biochemical and molecular responses to long-term salinity challenges in northern quahogs Mercenaria mercenaria

Salinity is a key environmental factor for aquatic organisms for survival, development, distribution, and physiological performance. Salinity fluctuation occurs often in estuary and coastal zones due to weather, tide, and freshwater inflow and thus heavily affects coastal marine aquaculture. The northern quahog Mercenaria mercenaria is an important aquaculture species along the Atlantic coast in the US, but information on the effects of salinity stress on physiological, immunological, and molecular responses is still scarce. The goal of this study was to investigate cellular and molecular responses through challenges of long-term hypo- and hyper-salinities in northern quahogs. The objectives were to: 1) measure the survival of market-sized quahogs under a three-month salinity challenge at 15 (hyposalinity), 25 (control), and 35 ppt (hypersalinity); 2) determine cellular changes of hemocytes through analysis of immune functions; 3) determine changes of the total free amino acid concentration in gills, and 4) evaluate the molecular responses in gills using RNAseq technology with qPCR verification. After a three-month salinity challenge, no mortality was observed, and increases in body weight were identified with a significantly higher increase in the hypersalinity group. Northern quahogs equilibrated their hemolymph osmolality with the ambient seawater and were verified to be osmoconformers. Significant differences were observed in total hemocyte concentration, lysosomal presence, ROS production, and phagocytic rate, but no differences were found in cell viability. The total free amino acid concentration within gills was positively correlated to water salinity, indicating amino acids were critical organic osmolytes. The transcriptome of gills using RNAseq revealed differential expression genes (DEG) encoding amino acid transporters (SLC6A3, SLC6A6, SLC6A13, SLC25A38), ion channel proteins (T38B1, GluCl, ATP2C1), and water channel protein (AQP8) in hyposalinity or/and hypersalinity groups, indicating these genes play critical roles in intracellular isosmotic regulation. Overall, the findings in this study provided new insights into osmoregulation in northern quahogs.

Leeches Baicalobdella torquata feed on hemolymph but have a low effect on the cellular immune response of amphipod Eulimnogammarus verrucosus from Lake Baikal.

Lake Baikal is one of the largest and oldest freshwater reservoirs on the planet with a huge endemic diversity of amphipods (Amphipoda, Crustacea). These crustaceans have various symbiotic relationships, including the rarely described phenomenon of leech parasitism on amphipods. It is known that leeches feeding on hemolymph of crustacean hosts can influence their physiology, especially under stressful conditions. Here we show that leeches Baicalobdella torquata (Grube, 1871) found on gills of Eulimnogammarus verrucosus (Gerstfeldt, 1858), one of the most abundant amphipods in the Baikal littoral zone, indeed feed on the hemolymph of their host. However, the leech infection had no effect on immune parameters such as hemocyte concentration or phenoloxidase activity and also did not affect glycogen content. The intensity of hemocyte reaction to foreign bodies in a primary culture was identical between leech-free and leech-infected animals. Artificial infection with leeches also had only a subtle effect on the course of a model microbial infection in terms of hemocyte concentration and composition. Despite we cannot fully exclude deleterious effects of the parasites, our study indicates a low influence of a few leeches on E. verrucosus and shows that leech-infected amphipods can be used at least for some types of ecophysiological experiments.

Interaction of water salinity and titanium dioxide nanoparticle (TiO2) exposure in the Pacific oyster, Crassostrea gigas: immune and antioxidant system responses

Titanium dioxide nanoparticles (TiO2) are released into the natural waters in different ways and cause toxicity to aquatic life. Although various studies have reported the toxicity of nanoparticles for aquatic animals, there is limited information regarding the effects of environmental factors, especially salinity, on their toxicity. Simultaneous effects of TiNPs and water salinity were studied in the Pacific oyster, Crassostrea gigas. Oysters were exposed to sub-lethal concentrations of TiNPs (low concentration: 50 µg/l, middle concentration: 500 µg/l, high concentration: 5000 µg/l) at different water salinities (Extremely low salinity: 5 g/l, low salinity: 15 g/l, middle salinity: 25 g/l, high salinity (normal salinity): 35 g/l) for 96 h. After nano-TiO2 exposure, the immune components in hemolymph (total hemocyte concentration [THC], phagocytosis), antioxidant enzyme activities in digestive gland (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase), oxidation stress index (malondialdehyde) in gills and the expression of immune-related genes (Vav-3 protein, cofilin, Galectin 4, pro-Cathepsin L, Rho protein, Ficolin, LPS/-1,3-glucan protein, Lysozyme, Defensin) in hemocytes were measured. The THC, phagocytosis, antioxidant enzyme activities, and immune-related gene expressions mostly decreased at salinities of 5 and 15 g/l in response to 500–5000 µg/l nano-TiO2 compared to non-exposed oysters and 50 µg/l nano-TiO2 group. An increase in water salinity up to 35 g/l mitigated the depressing effects of the nano-TiO2 on the immune and antioxidant components of the oysters. Exposure of oysters to 500–5000 µg/l nano-TiO2-induced oxidative stress at salinities of 5 and 15, since malondialdehyde levels were higher in these treatments compared to control and those in 50 µg/l nano-TiO2 group. The increase of salinity up to 35 g/l ameliorated the TiO2-induced oxidative stress. The results of the present study showed that the toxic impacts of nano-TiO2 can be ameliorated in the Pacific oyster with increase in water salinity, as the immune components, antioxidant enzyme activities recovered and oxidative stress mitigated when the salinity increased up to 35 g/l.

Differential responses of selectively bred mussels (Perna canaliculus) to heat stress-survival, immunology, gene expression and microbiome diversity.

New Zealand's green-lipped mussel (Perna canaliculus) is an ecologically and economically important species. Marine heatwaves are increasing in frequency around NZ's coastline, and these events are correlated with increased stress and mortality of some aquaculture species. This study aimed to identify general biomarkers of heat stress in P. canaliculus and to assess whether responses differed between genetically distinct selectively bred mussels. We exposed three families of selectively bred mussels (families A, B and C) to three seawater temperature regimes in the laboratory: 1) a "control" treatment (ambient 12°C), 2) a 26°C heat challenge with a subsequent recovery period, and 3) a sustained 26°C heat challenge with no recovery. We investigated whether the survival, immune response (hemocyte concentration and viability, oxidative stress and total antioxidant capacity), hemocyte gene expression and gill microbiome differed between the families during the temperature challenges. In the sustained heat-stress treatment, family A had the highest survival rate (42% compared with 25% and 5% for families C and B, respectively). Gene expression levels significantly shifted during thermal stress and differed between families, with family A more dissimilar than families B and C. Family C had substantially more genes impacted by temperature treatment and timepoint than the other families, while family B had very little genes/pathways that responded to thermal stress. Genes related to heat shock proteins and immune responses (e.g., AIF1, CTSC, TOLL8, CASP9, FNTA, AHCY, CRYAB, PPIF) were upregulated in all families during heat stress. Microbiome species-richness differed between families before and during heat-stress, with family A having a distinctly different microbiome flora than the other families. Microbial diversity changed similarly in all families exposed to prolonged heat-stress, with species of Vibrio and Campylobacter increasing in these mussels. Our study highlights the use of non-lethal sampling of hemocytes as a diagnostic tool to explore the immune response and gene expression of selectively bred mussels, to predict their response to ocean warming. This approach can identify potential thermotolerant candidates for further selective breeding, which may increase the resilience of the mussel aquaculture industry in a warming ocean.

Modeling seasonal immune dynamics of honey bee (Apis mellifera L.) response to injection of heat-killed Serratia marcescens.

The honey bee, Apis mellifera L., is one of the main pollinators worldwide. In a temperate climate, seasonality affects the life span, behavior, physiology, and immunity of honey bees. In consequence, it impacts their interaction with pathogens and parasites. In this study, we used Bayesian statistics and modeling to examine the immune response dynamics of summer and winter honey bee workers after injection with the heat-killed bacteria Serratia marcescens, an opportunistic honey bee pathogen. We investigated the humoral and cellular immune response at the transcriptional and functional levels using qPCR of selected immune genes, antimicrobial activity assay, and flow cytometric analysis of hemocyte concentration. Our data demonstrate increased antimicrobial activity at transcriptional and functional levels in summer and winter workers after injection, with a stronger immune response in winter bees. On the other hand, an increase in hemocyte concentration was observed only in the summer bee population. Our results indicate that the summer population mounts a cellular response when challenged with heat-killed S. marcescens, while winter honey bees predominantly rely on humoral immune reactions. We created a model describing the honey bee immune response dynamics to bacteria-derived components by applying Bayesian statistics to our data. This model can be employed in further research and facilitate the investigating of the honey bee immune system and its response to pathogens.

Heat stress has immediate and persistent effects on immunity and development of Tenebrio molitor

Abstract The yellow mealworm (Tenebrio molitor) is a promising insect species for mass-rearing for the production of feed and food. In mass-production systems, insects may be exposed to abiotic stressors such as heat stress as well as potentially lethal pathogens. To ensure mass-reared T. molitor populations are healthy and productive there is a need to understand both the risks, and potential benefits of heat stress, on the fitness of insects and their susceptibility to pathogens. In this study, we investigated the effects of a short (2 h) or a long (14 h) heat stress (38 °C) exposure on the susceptibility and the immune responses of T. molitor larvae exposed to a fungal pathogen (Metarhizium brunneum). Larvae were exposed to the pathogen either immediately or five days after the heat stress treatments. The development of heat stressed larvae and their offspring was also assessed. A short heat stress immediately before exposure to M. brunneum increased the survival probability of T. molitor larvae, which correlated with increased antibacterial activity in the hemolymph. The exposure of larvae to short, or long heat stresses five days before pathogen exposure did not affect their survival, despite a temporary lowered body mass gain of heat stressed larvae. However, heat stressed larvae showed decreased hemocyte concentrations when exposed to M. brunneum. We also found an increased body weight in larval offspring of females that had been exposed to a short heat stress as larvae themselves. These findings demonstrate the importance of understanding the effects of heat stress in the long-term. The beneficial effects of heat stress on pathogen susceptibility in T. molitor and the negative effects on body mass gain are only transient, whereas negative effects on immune response (hemocyte concentrations) persist over an extended period.

Viability Analysis of Biomphalaria glabrata Hemocytes During Schistosoma mansoni Infection And Glyphosate-Based Herbicide Exposure

Pesticides are chemical agents that have a range of harmful effects on human health and the environment. They can cause drastic changes in natural communities, such as macroinvertebrates, plankton and fish. Snails, including Biomphalaria glabrata, are often present in aquatic communities and have multiple roles in limnic ecosystems. B. glabrata is an intermediate host of several species of helminths of medical and veterinary importance, such as Schistosoma mansoni, the etiological agent of schistosomiasis. The original Roundup® herbicide can affect snails and directly affects S. mansoni hemocytes, cells that act in the snails’ immune defense. Here we analyzed the effect of herbicide exposure on B. glabrata hemocytes divided into four groups: control group, infected-only group, treated-only group and infected+treated group. For this, flow cytometry and Neubauer-chamber counting were used to determine the morphology, viability and lectin expression profiles of hemocytes. We observed that the group infected by S. mansoni and treated with herbicide had a higher concentration of dead hemocytes in relation to the other groups. The treated group showed similar results to the control group, suggesting that the herbicide (Roundup™) alone does not interfere with the snails’ immune system. Regarding cellular-morphological-characterization analysis, hyalinocytes were the cells most commonly found in all groups studied. These findings suggest that S. mansoni infection and exposure to pesticides directly the immune system of the snails, stimulating the production of hemocytes, especially hyalinocytes, which have a high phagocytic power to quell the infection, but with toxic effects on the snail.

Effect of endophytic entomopathogenic fungal conidia and blastospores induced in maize plants by seed inoculation on Spodoptera frugiperda immune response and mortality

Abstract. Sari JMP, Herlinda S, Suwandi S, Elfita. 2023. Effect of endophytic entomopathogenic fungal conidia and blastospores induced in maize plants by seed inoculation on Spodoptera frugiperda immune response and mortality. Biodiversitas 24: 5709-5717. Endophytic entomopathogenic fungi (EPF) can produce conidia and blastospores; however, the pathogenicity of conidia and blastospores inoculated in plants on Spodoptera frugiperda larvae is limited. This research aimed to detect the effect of the endophytic entomopathogenic fungal conidia and blastospores induced in maize plants by seed inoculation on S. frugiperda's immune response and mortality. A total of 10 isolates of endophytic EPF were used in this experiment. The study revealed that 9 days after treatments, S. frugiperda larvae consuming maize leaves inoculated with blastospores of endophytic entomopathogenic fungi; their hemocyte concentration did not show any significant differences between the fungal treatments and control. However, 1 up to 7 days after treatments, the concentration significantly differed from the control. Furthermore, B. bassiana JgSPK, JaGiP, and JaSpkPGA(2) isolates tended to be the most pathogenic compared to other isolates. Feeding on leaves colonized by endophytic EPF could reduce the larval and pupal weight of S. frugiperda. The percentage of S. frugiperda non-emergence pupae from larvae-eating maize leaves colonized with B. bassiana JgSPK and JaGiP isolates was significantly higher than other fungal treatments and the control. The endophytic entomopathogenic fungal conidia and blastospores inoculated in maize plants by seed inoculation have a lethal effect on S. frugiperda larvae. However, exposure to conidia and blastospores did not reduce or increase the hemocyte concentration in the larvae hemolymph of S. frugiperda.

Increased pupal temperature has reversible effects on thermal performance and irreversible effects on immune system and fecundity in adult ladybirds

The environmental conditions an organism encounters during development vary in their lasting impact on adult phenotypes. In the context of ongoing climate change, it is particularly relevant to understand how high developmental temperatures can impact adult traits, and whether these effects persist or diminish during adulthood. Here, we assessed the effects of pupal temperature (17 °C – normal temperature, 26 °C – increased temperature, or 35 °C – heat wave) on adult Harmonia axyridis thermal stress tolerance, immune function, starvation resistance, and fecundity. The temperature during pupation significantly affected all investigated traits in fresh adults. Heat acclimation decreased adult haemocyte concentration, cold tolerance, and total egg production, and had a positive effect on heat tolerance and starvation resistance. The negative effects of heat acclimation on cold tolerance diminished after seven days. In contrast, heat acclimation had a lasting positive effect on adult heat tolerance. Our results provide a broad assessment of the effects of developmental thermal acclimation on H. axyridis adult phenotypes. The relative plasticity of several adult traits after thermal acclimation may be consequential for the future geographic distribution and local performance of various insect species.

The Fungus Nosema ceranae and a Sublethal Dose of the Neonicotinoid Insecticide Thiamethoxam Differentially Affected the Health and Immunity of Africanized Honey Bees.

Honey bees (Apis mellifera L.) are affected by different biotic and abiotic stressors, such as the fungus Nosema ceranae and neonicotinoid insecticides, that negatively impact their health. However, most studies so far conducted have focused on the effect of these stressors separately and in European honey bees. Therefore, this study was conducted to analyze the impact of both stressors, singly and in combination, on honey bees of African descent that have demonstrated resistance to parasites and pesticides. Africanized honey bees (AHBs, Apis mellifera scutellata Lepeletier) were inoculated with N. ceranae (1 × 105 spores/bee) and/or chronically exposed for 18 days to a sublethal dose of thiamethoxam (0.025 ng/bee) to evaluate their single and combined effects on food consumption, survivorship, N. ceranae infection, and immunity at the cellular and humoral levels. No significant effects by any of the stressors were found for food consumption. However, thiamethoxam was the main stressor associated to a significant decrease in AHB survivorship, whereas N. ceranae was the main stressor affecting their humoral immune response by upregulating the expression of the gene AmHym-1. Additionally, both stressors, separately and combined, significantly decreased the concentration of haemocytes in the haemolymph of the bees. These findings indicate that N. ceranae and thiamethoxam differentially affect the lifespan and immunity of AHBs and do not seem to have synergistic effects when AHBs are simultaneously exposed to both stressors.

Age-Related and Seasonal Dynamics of Hemocyte Population in the Mussel <i>Crenomytilus grayanus</i> (Dunker, 1853)

The cellular composition of the hemolymph of the mussel Crenomytilus grayanus is represented by hemoblasts, hyalinocytes, basophilic, neutrophilic and acidophilic granulocytes with a significant predominance of the latter. With the age of the mollusk, the average values of the size and granularity of hemocytes increased due to accumulation of granulocytes in the hemolymph (up to 100% of all cells) and their mature highly differentiated acidophilic forms (up to 99% of all cells); this indicates a decrease in the rate of renewal of the cell population. During the autumn period of sexual inertia, the number of cells in the hemolymph of mussels decreased with increasing age of mollusks but without any correlation with the shell size. However, in summer, during the spawning period, the concentration of hemocytes either did not change or increased with increasing body size, directly correlating with the number of young basophilic granulocytes. This correlation may indicate either a relationship between the number of hemocytes and the ability of mussels to reach large sizes, or a higher potency in aged mussels for emergency mobilization of the immune system in the summer season through rapid replenishment of the pool of circulating cells with subsequent redistribution of young and mature cell forms between hemolymph and the internal organs.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity

BackgroundMosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated.MethodsLarvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR.ResultsPropagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF.ConclusionBy 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.Graphical

Microplastics can aggravate the impact of ocean acidification on the health of mussels: Insights from physiological performance, immunity and byssus properties

Ocean acidification may increase the risk of disease outbreaks that would challenge the future persistence of marine organisms if their immune system and capacity to produce vital structures for survival (e.g., byssus threads produced by bivalves) are compromised by acidified seawater. These potential adverse effects may be exacerbated by microplastic pollution, which is forecast to co-occur with ocean acidification in the future. Thus, we evaluated the impact of ocean acidification and microplastics on the health of a mussel species (Mytilus coruscus) by assessing its physiological performance, immunity and byssus properties. We found that ocean acidification and microplastics not only reduced hemocyte concentration and viability due to elevated oxidative stress, but also undermined phagocytic activity of hemocytes due to lowered energy budget of mussels, which was in turn caused by the reduced feeding performance and energy assimilation. Byssus quality (strength and extensibility) and production were also reduced by ocean acidification and microplastics. To increase the chance of survival with these stressors, the mussels prioritized the synthesis of some byssus proteins (Mfp-4 and Mfp-5) to help maintain adhesion to substrata. Nevertheless, our findings suggest that co-occurrence of ocean acidification and microplastic pollution would increase the susceptibility of bivalves to infectious diseases and dislodgement risk, thereby threatening their survival and undermining their ecological contributions to the community.

Fungal ectoparasites increase winter mortality of ladybird hosts despite limited effects on their immune system.

Winter represents a challenging period for insects inhabiting temperate regions. A plethora of studies have investigated how environmental conditions such as temperature affect insect overwintering success. However, only a few studies have focused on biotic factors and the mechanisms affecting the overwintering performance of insects. Here, we investigated the effects of the parasitic fungus Hesperomyces virescens on the overwintering performance and immune system functioning of the invasive ladybird Harmonia axyridis. Winter survival was significantly lower for infected than for uninfected ladybirds. Body mass loss during overwintering tends to be higher for infected individuals compared to uninfected ones and for larger ladybirds. In addition, parasitic infection reduced post-winter longevity without food in male but not female ladybirds. Total haemocyte and protein concentration as well as antimicrobial activity against Escherichia coli significantly decreased during ladybird overwintering. However, haemolymph parameters were only poorly affected by Hesperomyces infection, with the exception of antimicrobial activity against E. coli that tended to be higher in infected ladybirds. Interestingly, none of the pre-winter haemolymph parameters were good predictors of ladybird winter survival. Overall, our results indicate that energy exhaustion unrelated to immune system challenge is the most probable explanation for increased overwintering mortality in infected ladybirds.

BASELINE HEMATOLOGIC AND BIOCHEMICAL VALUES AND CORRELATIONS TO ENVIRONMENTAL PARAMETERS IN MANAGED JAPANESE SPIDER CRABS (MACROCHEIRA KAEMPFERI)

Although the evaluation of hematologic and biochemical parameters is a well-established diagnostic tool in vertebrate medicine, comprehensive understanding of these parameters in invertebrate species is lacking. This study provides baseline hemocyte concentrations and biochemistry values for a population of managed Japanese spider crabs (JSC; Macrocheira kaempferi) housed at six different public aquariums. The methodology for obtaining diagnostic hemolymph samples is described. Distinct hemocyte types were identified, including hyaline cells, semigranulocytes, and granulocytes, with hyaline cells as the predominant type. Correlates to exam findings and environmental parameters were evaluated and included higher absolute semigranulocyte counts (r = 0.65, P = 0.020) and triglyceride levels (r = 0.44, P = 0.014) in JSC with exoskeletal lesions; higher total protein (mean = 5.93 g/dl, P = 0.028), cholesterol (median = 18.5 mg/dl, P = 0.018), triglyceride (median = 15.5 mg/dl, P = 0.002), and amylase (median = 243 U/L, P = 0.013) in nonmolting JSC compared with JSC that have previously molted since acquisition (total protein mean = 4.83 g/dl, cholesterol median = 14 mg/dl, triglyceride median = 6.4 mg/dl, and amylase median = 131 U/L); and lower relative and absolute granulocyte counts (mean = 8.83% P = 0.030, median = 1,162 cells/µl P = 0.006, respectively) and higher albumin (median = 1.35 g/dl, P = 0.031) in JSC housed at facilities that used ozone sterilization. The data presented serve as a foundation for understanding basic clinical parameters in JSC hemolymph, as well as the potential influence of environmental stressors on those parameters.

Use of an exotic host plant shifts immunity, chemical defense, and viral burden in wild populations of a specialist insect herbivore.

Defense against natural enemies constitutes an important driver of herbivore host range evolution in the wild. Populations of the Baltimore checkerspot butterfly, Euphydryas phaeton (Nymphalidae), have recently incorporated an exotic plant, Plantago lanceolata (Plantaginaceae), into their dietary range. To understand the tritrophic consequences of utilizing this exotic host plant, we examined immune performance, chemical defense, and interactions with a natural entomopathogen (Junonia coenia densovirus, Parvoviridae) across wild populations of this specialist herbivore. We measured three immune parameters, sequestration of defensive iridoid glycosides (IGs), and viral infection load in field‐collected caterpillars using either P. lanceolata or a native plant, Chelone glabra (Plantaginaceae). We found that larvae using the exotic plant exhibited reduced immunocompetence, compositional differences in IG sequestration, and higher in situ viral burdens compared to those using the native plant. On both host plants, high IG sequestration was associated with reduced hemocyte concentration in the larval hemolymph, providing the first evidence of incompatibility between sequestered chemical defenses and the immune response (i.e., the “vulnerable host” hypothesis) from a field‐based study. However, despite this negative relationship between IG sequestration and cellular immunity, caterpillars with greater sequestration harbored lower viral loads. While survival of virus‐infected individuals decreased with increasing viral burden, it ultimately did not differ between the exotic and native plants. These results provide evidence that: (1) phytochemical sequestration may contribute to defense against pathogens even when immunity is compromised and (2) herbivore persistence on exotic plant species may be facilitated by sequestration and its role in defense against natural enemies.

Effect of plant phenolic compounds on the hemocyte concentration and antioxidant enzyme activity in Hyphantria cunea (Drury, 1773) (Lepidoptera: Arctiidae) larvae infected by Hyphantria cunea granulovirus

The aim of this study was to determine the effects of phenolic substances in different plants (mulberry, apple, walnut and plum) on hemocyte counts and antioxidant enzyme activities of Hyphantria cunea larvae infected with Hyphantria cunea granulovirus (HycuGV) and uninfected. In the study, the phenolic contents of plant materials were firstly determined. Then, the effects of these ingredients on hemocyte counts and antioxidant enzyme activities of infected and uninfected larvae were determined. As a result of the study, the hemocyte counts of all groups increased with the infection was determined. In enzyme activity, the phenoloxidase (PO) activities of larvae fed on apple leaves and plum leaves containing rosmarinic acid and protocatechuic acid increased with infection was determined. The amounts of malondialdehyde (MDA) decreased in all groups as a result of viral infection. While the highest superoxide dismutase (SOD) and catalase (CAT) activities among both infected and uninfected larvae were in the plum groups with the highest amount of chlorogenic acid, the lowest glutathione peroxidase (GSH-Px) activities were also in these groups. All this showed that different phenolic contents of host plants affected the hemocyte counts and antioxidant enzyme activities of H. cunea larvae.

Physiological and antioxidant response of Litopenaeus vannamei against Vibrio parahaemolyticus infection after feeding supplemented diets containing Dunaliella sp. flour and β-glucans

The presence of pathogen agents in shrimp farming is the main obstacle for successful aquaculture. Vibrio species are naturally part of water because they play an important role as opportunistic bacteria. Vibrio parahaemolyticus was identified as the causative agent of the Early Mortality Syndrome in 2009, causing the loss of shrimp farming worldwide. Dunaliella sp. flour has been tested against Vibrio infection proving to be an effective prophylactic method that decreases mortality and improves physiological and immune response in Litopenaeus vannamei. Juvenile shrimp were exposed to 2% Dunaliella sp. flour and commercial 1.1% β -glucan diet provided every other day for 15 days and a posterior infection with V. parahaemolyticus (1 × 106 CFU/mL). To evaluate shrimp stress status, some parameters as glucose, lactate, cholesterol, triglycerides, relative superoxide dismutase (SOD) gene expression and circulating hemocytes were analyzed in hemolymph at zero and seven days before infection and at 0, 24, and 48 h post-infection. L. vannamei fed with Dunaliella sp. showed 93% and β -glucan 87% survival, compared with 79% in the infected control group. Additionally, Dunaliella sp. improved hemocyte and lipid concentrations compared to β -glucan while both immunostimulants showed an increase in SOD response against bacteria. The addition of 2% Dunaliella sp. every other day in L. vannamei diet enhanced stress response against V. parahaemolyticus infection.

No effect of abscisic and p-coumaric acids as food supplements and stimulants of the immunological system of Africanized hybrids of Apis mellifera

Bee colonies determined as Apis mellifera scutellata haplotype A1 were administered with a food supplement of sugar syrup mixed with p-coumaric acid (CUM), to test its supposed antioxidant effects; abscisic acid (ABA), to test whether it promotes innate immunity in bees, the combination of both (ABACUM), and syrup as a control. During three months, the number of cells with brood, honey and pollen reserves, as well as the concentration of hemocytes in worker bees were counted. Besides, Varroa destructor and Nosema ceranae were monitored. At the concentrations used, ABA and CUM had no effect on oviposition; however, there was a significant increase in the number of cells with brood or with honey and pollen reserves between the beginning and the end of the study. Regarding the monitoring of V. destructor, there were no significant differences between treatments; this mite did not cause obvious damage, seemingly because the experimental (Africanized) bees are considered tolerant to its infestation. There were no significant differences between treatments in infection by N. ceranae; however, there was a significant decrease in intensity, prevalence, and abundance of spores between the beginning and the end of the study, which was associated with the fact that the bees collected nectar and this resulted in the development of colonies. The number of hemocytes did not show a significant difference between treatments, but there was a significant increase between the first and the last evaluation. Neither ABA, CUM or ABACUM produced the predicted effects on the experimental bees.

Flow cytometric validation of a commercial kit to assess the concentration and viability of bivalve hemocytes

• A protocol to measure the concentration and viability of Perna canaliculus hemocytes was validated. • The validated assay, using a simple user interface flow cytometer, was compared to 3 other assays. • Using physiologically-compatible media with commercially-available assay kits is important.