Hemolymph Proteins Research Articles

Insights into larval development and protein biochemical alterations of Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae) following Beauveria bassiana and Solanum lycopersicum treatments

BackgroundThe polyphagous notorious pest, black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), cause significant production losses due to its distinctive feeding and hiding behavior, making it particularly challenging to control it with conventional methods. Therefore, sustainable agriculture demands more effective and environmentally safe pest control solutions. This study aimed to investigate the toxicity of two insecticide alternatives, the entomopathogenic fungus (EPF) Beauveria bassiana and Solanum lycopersicum extract (Tomato plant crude extract, TPCE), using two bioassay methods: the poisoned bait method and the leaf dipping method. In addition, the impact of these biological tools on larval development and protein profiles was evaluated.ResultsThe bait application of both tested materials exhibited higher toxicity than the leaf dipping method, as indicated by the toxicity index. The LC50 values for B. bassiana were 1.6 × 10⁸ and 1.8 × 10⁶ conidia ml−1 using the leaf dipping method and poisoned baits method, respectively. For TPCE, the LC50 values were 4.35 and 1.51 mg ml−1 for the same methods, respectively. In addition, sublethal concentrations of both materials altered the larval and pupal durations. B. bassiana significantly reduced the concentration of larval hemolymph protein. A maximum of 12 protein bands in the control sample, with molecular weights (Mw) ranging between 35 and 120 kDa, were detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In B. bassiana-treated larvae, ten bands were detected with Mw ranging from 35 to 120 kDa. At least seven bands were detected in TPCE-treated larvae, with Mw ranging from 35 to 97 kDa.ConclusionsThe findings of this study can be integrated into management programs for A. ipsilon. In addition, the availability of B. bassiana and TPCE in Egypt and their cost-effectiveness as insecticide alternatives support their use in the management programs of this critical pest. These methods are particularly effective when applied in bait form.Graphical

Effect of abamectin on haemolymph and ovarian proteins during the reproductive cycle of female Argas (Persicargas) arboreus (Acari: Argasidae)

AbstractThe avian tick Argas (Persicargas) arboreus is a vector of pathogens infecting the nestlings of the agriculturally beneficial bird, the buff-backed heron in Egypt. The topical treatment of the fed-mated (engorged) female A. (P.) arboreus with a sublethal dose of abamectin (10 µl 10 ppm/female) on the 3rd day after feeding (daf), reduced the concentration of total protein in the haemolymph (HL) by 13–66%, in the ovary by 30–54%, and in freshly deposited eggs by 11–16%, compared to untreated females during vitellogenesis and oviposition. However, the treatment did not prevent vitellogenesis and oviposition but reduced the number of mature oocytes (stages III–V) and deposited eggs. Additionally, it delayed the onset and slowed the progress of each process in the treated females. The application of abamectin at the onset of vitellogenesis (on the 3rd daf) in normal female and the prominent decrease inHL total protein concentration in treated female on the 4th daf pointed to a probable interference of abamectin with the release of the vitellogenesis-inducing factor (VIF) and/or other subsequent hormone(s) known to regulate vitellogenesis in this argasid tick species. Graphical abstract

Differential expression of hemolymph proteins in wild bumblebees provides insights into species‐specific impacts of heat stress

AbstractWildlife faces an increasing threat from extreme climatic events, such as heatwaves, which can have a severe impact on various species, including crucial pollinators like bumblebees. Bumblebees are cold‐adapted and heterothermic, possessing the ability to regulate their internal temperature. The impact of heat stress seems species specific in bumblebees. While most species are impacted, some bumblebee species manage to survive, potentially by employing physiological mechanisms, including the modulation of their protein profile (e.g. Heat Shock Proteins). However, there is limited understanding of how their protein profiles are associated with heat exposure. In this study, we examined the global variation in the protein profile of males from two bumblebee species sampled in the wild: the heat‐tolerant Bombus terrestris and the heat‐sensitive Bombus magnus. After subjecting them to heat stupor at 40°C in controlled condition, it was observed that nearly all B. terrestris survived the stress, while over 50% of B. magnus individuals succumbed to the heat exposure. Through off‐gel bottom‐up proteomics and LC–MS/MS analysis of the hemolymph proteome, we identified 164 proteins in both species with a large part of differentially expressed proteins after heat exposure. Additionally, quantitative analysis of fat bodies revealed that the relative mass was stable in B. terrestris, while it was significantly lower in B. magnus exposed to heat stress. Our data suggest that compared with B. magnus, B. terrestris displays a higher adaptability of its hemolymph proteome in response to heat stress. This adaptability could be a key factor contributing to the high physiological resistance of B. terrestris and its ability to adapt to new, stressful environments expected due to climate change. Understanding these mechanisms of protein regulation in bumblebees could provide valuable insights into their resilience and vulnerability facing environmental stresses.

Effect of heat stress on Apis mellifera intermissa and Apis mellifera sahariensis in laboratory controlled conditions: a physiological approach

The effects of heat on honey bees have received attention in many subspecies. Nevertheless, some subspecies still remain unexplored. In this study, we investigate for the first time the effect of heat on two subspecies of honey bees endemic to Algeria, Apis mellifera intermissa which is adapted to the Mediterranean climate and Apis mellifera sahariensis, which is adapted to the hot climate of southwest Algeria. We tested the hypothesis that sahariensis is more tolerant to heat stress than intermissa. In two in-vitro tests, we reared workers of both subspecies at 4 temperatures (34 °C, 38 °C, 39 °C, and 40 °C). Then, we determined the hemolymph protein (HP) concentration, hypopharyngeal gland development (HPG), ovary development and survival. We found that the sahariensis had HPG more developed and less affected by heat than intermissa. Conversely, although intermissa had more (HP) at 34 °C, we found a significant increase in (HP) at 38 °C, 39 C° and 40 °C in intermissa than sahariensis. Although ovary development is greater in intermissa than sahariensis at 34 °C and greater at 34 °C than 38 °C, 39 °C and 40 °C, we did not record differences in ovary development between the subspecies at 38 °C, 39 °C, and 40 °C. Finally, in all conditions, sahariensis had a higher survival than intermissa.

Biopesticidal Potential of Lippia javanica (Burm. F) Spreng. Leaf Extracts and their Fractions against Spodoptera litura (Fab.)

Three organic solvent extracts of Lippia javanica leaves were screened for their antifeedant and larvicidal activities against the 4th instar larvae of Spodoptera litura at 5% concentration. The maximum antifeedant and larvicidal activities were recorded in ethyl acetate extract (76.57%) for antifeedant and (90.40%) for larvicidal activity, followed by chloroform and hexane extracts. Ten fractions were obtained from the ethyl acetate extract of L. javanica by using different combinations of hexane and ethyl acetate as the mobile phase through column chromatography. The fractions were screened at 1000 ppm concentrations for antifeedant and larvicidal activities. Fraction 10 (71.75%) was found to be the most effective one, followed by fraction 4 (61.32%) and fraction 7 (60.58%) for antifeedant acitivity. At a 1000 ppm concentration, fraction 3 exhibited the highest larvicidal activity (79.20%) against S. litura, followed by fraction 1 (76.0%), while fractions 5 and 6 have shown equal activity. Quantitative protein analysis revealed that treatment with the eighth fraction reduced the haemolymph protein drastically (1.52 mg/mL) compared to control (2.84 mg/mL) and other fractions. In the reference control azadirachtin treatment, the haemolymph protein quantity was found to be nearly the same (1.4 mg/mL) as that of the eighth fraction. Treatment with the first and second fractions resulted in increased haemolymph protein (8 and 7.6 mg/mL, respectively). The bio-efficacy of L. javanica in pest management is discussed.

Field Application of an Innovative Approach to Assess Honeybee Health and Nutritional Status.

Environment, forage quality, management practices, pathogens, and pesticides influence honeybee responses to stressors. This study proposes an innovative approach to assess colony health and performance using molecular diagnostic tools by correlating hemolymph proteins with common measures of colony strength, prevalent honeybee pathogens (Varroa destructor and Nosema spp.), and essential trace elements (iron, zinc and copper). Colonies were selected from four apiaries located in different environmental and foraging conditions in the province of Bologna (Italy). Hemolymph samples were taken from June to October 2019. The Varroa infestation of the colonies was estimated by assessing the natural mortality of the mites, while the bees were tested for Nosema spp. spores using a microscopic method. Hemolymph proteins were quantified and separated using SDS-PAGE, and colony performance was assessed by determining adult bees, total brood, honey, and pollen reserves. The biomarkers measured proved to be useful for monitoring changes in performance and trophic conditions during summer and early autumn. Significant correlations were found between hemolymph proteins and colony performance measures. A positive correlation between pollen reserves, vitellogenin, and hexamerin 70a highlights the importance of these proteins for successful overwintering. In October, Varroa infestation was negatively correlated with total proteins, vitellogenin, apolipophorin II, transferrin, and hexamerin 70a, with negative implications for overwintering; furthermore, Varroa infestation was also negatively correlated with iron content, potentially affecting iron homeostasis.

What are the main proteins in the hemolymph of Haemaphysalis flava ticks?

Haemaphysalis flava is a notorious parasite for humans and animals worldwide. The organs of H. flava are bathed in hemolymph, which is a freely circulating fluid. Nutrients, immune factors, and waste can be transported to any part of the body via hemolymph. The main soluble components in hemolymph are proteins. However, knowledge of the H. flava proteome is limited. The hemolymph was collected from fully engorged H. flava ticks by leg amputation. Hemolymph proteins were examined by both blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from the gels were further identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two bands (380 and 520 kDa) were separated from tick hemolymph by BN-PAGE and were further separated into four bands (105, 120, 130, and 360 kDa) by SDS-PAGE. LC-MS/MS revealed that seven tick proteins and 13 host proteins were present in the four bands. These tick proteins mainly belonged to the vitellogenin (Vg) family and the α-macroglobulin family members. In silico structural analysis showed that these Vg family members all had common conserved domains, including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD), and the domain of unknown function (DUF). Additionally, two of the Vg family proteins were determined to belong to the carrier protein (CP) by analyzing the unique N-terminal amino acid sequences and the cleaving sites. These findings suggest that the Vg family proteins and α-macroglobulin are the primary constituents of the hemolymph in the form of protein complexes. Our results provide a valuable resource for further functional investigations of H. flava hemolymph effectors and may be useful in tick management.

Assessment of Amphibola crenata as a bioindicator of estuarine trace element pollution using biochemical and physiological endpoints

To assess its utility as a bioindicator of estuarine contamination, Amphibola crenata, a pulmonate mud snail, was collected from 17 estuaries in New Zealand. Whole-body soft tissue trace element concentrations were measured via quadrupole inductively coupled plasma mass spectrophotometer (Q-ICP-MS) and were found to be significantly positively correlated with sediment trace element profiles for arsenic, copper and lead. Snails from polluted estuaries generally displayed higher ammonia excretion rates, elevated whole-body soft tissue catalase activity and lipid peroxidation compared to snails from reference sites. Across all sites haemolymph glucose was positively correlated with the tissue burdens of arsenic, copper, nickel and zinc, while haemolymph protein was negatively correlated with arsenic, cadmium and zinc soft tissue concentrations, indicative of altered energy metabolism associated with trace element contamination. Overall, sites were distinguishable by application of the array of measures employed. Our findings suggest that gastropods have significant value as bioindicators of estuarine health.

Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals-Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph.

Insects are of great interest as novel sources of alternative proteins and biologically active compounds, primarily anticancer agents. Protein-rich insect larval hemolymph is a prospective candidate for pharmaceutical and food industry-related research. In this study, selected biochemical properties and cell toxicity of larval hemolymph from two mealworm species, Tenebrio molitor and Zophobas morio, were analyzed. Total proteins and carbohydrates, antioxidant capacity, and the level of lipid peroxidation were determined. Human cancer (U-87) and normometabolic (MRC-5) cells were treated with different concentrations of larval hemolymph proteins, and the effects on cell viability were assayed 24, 48, and 72 h after treatments. Z. morio hemolymph was shown to be richer in total proteins, showing a higher antioxidant capacity and lipid peroxidation level than T. molitor hemolymph, which was richer in total carbohydrates. Cytotoxicity assays showed that T. molitor and Z. morio hemolymphs differently affect the viability of U-87 and MRC-5 cells in cell type-, dose-, and time-dependent manners. Hemolymph from both species was more cytotoxic to U-87 cells than to MRC-5 cells, which was particularly prominent after 48 h. Additionally, a more potent cytotoxic effect of Z. morio hemolymph was observed on both cell lines, likely due to its higher antioxidant capacity, compared to T. molitor hemolymph.

Enhanced Recombinant Protein Expression in Insect Cells by Natural and Recombinant Components of Lepidoptera Hemolymph.

Prior research has established the anti-apoptotic effects in insect cell cultures of Bombyx mori (B. mori) hemolymph, as well as the heightened production yields of recombinant proteins facilitated by baculovirus vectors in insect cells cultivated in media supplemented with this hemolymph. In this study, we investigated the hemolymph of another Lepidoptera species, Trichoplusia ni (T. ni), and observed similar beneficial effects in insect cells cultivated in media supplemented with this natural substance. We observed enhancements in both production yield (approximately 1.5 times higher) and late-stage cell viabilities post-infection (30-40% higher). Storage-protein 2 from B. mori (SP2Bm) has previously been identified as one of the abundant hemolymph proteins potentially responsible for the beneficial effects observed after the use of B. mori hemolymph-supplemented cell culture media. By employing a dual baculovirus vector that co-expresses the SP2Bm protein alongside the GFP protein, we achieved a threefold increase in reporter protein production compared to a baculovirus vector expressing GFP alone. This study underscores the potential of hemolymph proteins sourced from various Lepidoptera species as biotechnological tools to augment baculovirus vector productivities, whether utilized as natural supplements in cell culture media or as hemolymph-derived recombinant proteins co-expressed by baculovirus vectors.

Bioaccumulation and sub-lethal physiological effects of metal mixtures on mussel, Mytilus edulis: Continuous exposure to a binary mixture of mercury and cadmium

In the natural ecosystem, aquatic organisms are exposed to a cocktail of chemicals that may result in toxicological responses differing from those of individual chemicals. In the present study, mussels were exposed using a semi-static and triplicated design to either control (no added metal), 50 µg l−1 (Hg alone), 50 µg l−1 (Cd alone), or 50 µg l−1 Hg plus 50 µg l−1 Cd (Hg + Cd) mixture for 14 days. Tissues were collected on days 0, 2, 4, 8, and 14 for metal analysis and sub-lethal responses using a suite of assays. Tissue metal concentrations were not significantly different in the single metal (Hg or Cd) compared to the Hg plus Cd mixture treatment for all tissues, apart from the gill of the Cd alone treatment. At the end of the experiment, the gill Cd concentration was significantly increased in the Hg plus Cd mixture compared to the Cd alone treatment, suggesting the influence of Hg on Cd uptake. The percentage increases of the Hg plus Cd mixture compared to the arithmetic sum of the individual metals were ( %): 20.2, 9.3, 25.1, 23.8, 10.7, and 12.4 for adductor muscle, digestive gland, gill, gonad, remaining soft tissue, and haemolymph, respectively. There were no observed treatment effects on total haemocyte count, haemolymph protein, or glucose concentration in the cell-free haemolymph. Neither was there any treatment effect on osmotic pressure, ions in the tissues, or in the cell-free haemolymph. At the end of the experiment, Hg-mediated oxidative damage, as an increase of thiobarbituric reactive substances (TBARS) and apparent depletion of total glutathione. This was observed in the gill and digestive gland of the Hg alone and Hg plus Cd mixture. Histopathology examination showed similar pathology in the Hg alone and the Hg plus Cd treatment. In conclusion, despite some oxidative stress and pathology during metal exposure, the accumulation of metals and effects on mussel health were similar between single exposures and a mixture of Hg plus Cd. In terms of risk assessment, regulations for the individual metals should suffice to protect against the mixture of Hg plus Cd, at least for adult M. edulis in full-strength seawater.

Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

The impact of Roundup® Original DI on the hemolymph metabolism and gill and hepatopancreas oxidative balance of Parastacus promatensis (Crustacea, Decapoda, Parastacidae).

In Brazil, glyphosate is present in more than 130 commercial formulations, and its toxic effects have already been tested in different species to understand its impact on biota Decapod crustaceans are widely used as experimental models due to their biology, sensitivity to pollutants, ease of collection, and maintenance under laboratory conditions. We evaluated the changes in metabolism (hemolymph) and oxidative balance markers (gill and hepatopancreas) of a crayfish (Parastacus promatensis) after exposure to Roundup® (active ingredient: glyphosate). The crayfish were captured in the Garapiá stream within the Center for Research and Conservation of Nature Pró-Mata, Brazil. We collected adult animals outside (fall) and during (spring) the breeding season. The animals were transported in buckets with cooled and aerated water from the collection site to the aquatic animal maintenance room at the university. After acclimatization, the animals were exposed to different concentrations of glyphosate (0, 65, 260, 520, and 780 µg/L). The results showed a significant variation in the hemolymph glucose, lactate, and protein levels. We observed variations in the tissue antioxidant enzymatic activity after exposure to glyphosate. Finally, the increase in oxidative damage required a high energy demand from the animals to maintain their fitness, which makes them more vulnerable to stress factors added to the habitat.

Comparative proteomic analysis of the hemolymph and salivary glands of Rhodnius prolixus and R. colombiensis reveals candidates associated with differential lytic activity against Trypanosoma cruzi Dm28c and T. cruzi Y.

Immune response of triatomines plays an important role in the success or failure of transmission of T. cruzi. Studies on parasite-vector interaction have shown the presence of trypanolytic factors and have been observed to be differentially expressed among triatomines, which affects the transmission of some T. cruzi strains or DTUs (Discrete Typing Units). Trypanolytic factors were detected in the hemolymph and saliva of R. prolixus against epimastigotes and trypomastigotes of the Y strain (T. cruzi II). To identify the components of the immune response that could be involved in this lytic activity, a comparative proteomic analysis was carried out, detecting 120 proteins in the hemolymph of R. prolixus and 107 in R. colombiensis. In salivary glands, 1103 proteins were detected in R. prolixus and 853 in R. colombiensis. A higher relative abundance of lysozyme, prolixin, nitrophorins, and serpin as immune response proteins was detected in the hemolymph of R. prolixus. Among the R. prolixus salivary proteins, a higher relative abundance of nitrophorins, lipocalins, and triabins was detected. The higher relative abundance of these immune factors in R. prolixus supports their participation in the lytic activity on Y strain (T. cruzi II), but not on Dm28c (T. cruzi I), which is resistant to lysis by hemolymph and salivary proteins of R. prolixus due to mechanisms of evading oxidative stress caused by immune factors. The lysis resistance observed in the Dm28c strain would be occurring at the DTU I level. T. cruzi I is the DTU with the greatest geographic distribution, from the south of the United States to central Chile and Argentina, a distribution that could be related to resistance to oxidative stress from vectors. Likewise, we can say that lysis against strain Y could occur at the level of DTU II and could be a determinant of the vector inability of these species to transmit T. cruzi II. Future proteomic and transcriptomic studies on vectors and the interactions of the intestinal microbiota with parasites will help to confirm the determinants of successful or failed vector transmission of T. cruzi DTUs in different parts of the Western Hemisphere.

Semi-purified Antimicrobial Proteins from Oyster Hemolymph Inhibit Pneumococcal Infection

Pneumococcal infections caused by Streptococcus pneumoniae are a leading cause of morbidity and mortality globally, particularly among children. The ability of S. pneumoniae to form enduring biofilms makes treatment inherently difficult, and options are further limited by emerging antibiotic resistance. The discovery of new antibiotics, particularly those with antibiofilm activity, is therefore increasingly important. Antimicrobial proteins and peptides (AMPs) from marine invertebrates are recognised as promising pharmacological leads. This study determined the in vitro antibacterial activity of hemolymph and unique protein fractions from an Australian oyster (Saccostrea glomerata) against multi-drug-resistant S. pneumoniae. We developed a successful method for hemolymph extraction and separation into 16 fractions by preparative HPLC. The strongest activity was observed in fraction 7: at 42 µg/mL protein, this fraction was bactericidal to S. pneumoniae and inhibited biofilm formation. Proteomic analysis showed that fraction 7 contained relatively high abundance of carbonic anhydrase, cofilin, cystatin B–like, and gelsolin-like proteins, while surrounding fractions, which showed lower or no antibacterial activity, contained these proteins in lower abundance or not at all. This work supports traditional medicinal uses of oysters and contributes to further research and development of novel hemolymph/AMP-based treatments for pneumococcal infections.

Effects of Spirulina platensis mediated TIO2NPS on biochemical analysis (Bombyx mori. L)

The treatment comprised S. platensis mediated TiO2NPs at different concentrations of 25, 50, 75, 100, 125 and 150ppm was diluted from the stock solution. The Double Hybrid silkworm (CSR6 ×CSR26) × (CSR2 × CSR 27) were fed with mulberry leaves treated with S. platensis mediated TiO2NPs.The application of mulberry leaves treated with S. platensis mediated TiO2NPs influenced silkworm economic parameters. The S. platensis mediated TiO2NPs at the concentration of 50 ppm treated mulberry leaves fed to silkworm which recorded increased Total haemolymph protein (8.62 and 8.63 mg/ml), Haemolymph carbohydrates(14.96 and 14.97 mg/ml) and Haemolymph lipids(19.99 and 20.00 mg/g)as compared to control.

Seasonal Changes in Hemolymph Protein Level and Hypopharyngeal Gland Size Depending on Age and In-Nest Location of Honeybee Workers

A honeybee colony, as a super-organism, is regulated through age-polyethism. A honeybee worker’s age is considered by means of a chronological and biological approach. The biological age is estimated with physiologically related biological markers, e.g., total hemolymph protein content (THP) and hypopharyngeal gland size (HGs), which also vary seasonally. Contemporary insights into the age-related spatial workers’ distribution within the hive nest space regarding biological age are insufficiently clarified. This study aimed to monitor changes in selected physiological markers during the entire season in relation to worker age and their spatial position in the hive nest. THP content and HG size analysis was performed in nine colonies for the entire season to compare the physiological markers within and among the groups of the workers whose ages were known and sampled in different hive parts. Seasonal impact on the biomarkers’ development was confirmed in known-age workers. In the case of HGs, this impact was the most apparent in 4- and 5-week-old workers. For THP, the seasonal impact was the most obvious in 2-week-old workers. The highest THP was found in 1- and 2-week-old workers during the entire season. Biologically younger workers of the same age were located predominantly in upper hive parts consistently throughout the year and vice versa. These workers showed significantly higher THP in comparison with those sampled below. Regarding the chronological age, the downwards, spatially shifting mechanism of workers within the hive nest while they aged was characterized. We recommend storage of diluted hemolymph samples up to one month before performing an assay if necessary. The physiological context, relation to division of labor and benefits for beekeeping practices are discussed.

Comparative hemolymph proteomic analyses of the freezing and resistance-freezing Ostrinia furnacalis (Guenée)

The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is one of the most harmful pests of maize in Asia. It poses a significant threat to maize production, causing economic losses due to its strong ecological adaptation. In this study, we compared and analyzed the hemolymph proteome between freezing and resistance-freezing O. furnacalis strains using two-dimensional gel electrophoresis to gain insights into the mechanisms of cold resistance. The results revealed that 300–400 hemolymph protein spots were common, with 24 spots showing differences between the two strains. Spectrometry analysis revealed 21 protein spots, including 17 upregulated spots and 4 downregulated ones. The expression of upregulation/downregulation proteins plays a crucial role in the metabolism, energy supply, and defense reaction of insects. Proteomics research not only provides a method for investigating protein expression patterns but also identifies numerous attractive candidates for further exploration.

Destruxin A inhibits the hemocytin-mediated hemolymph immunity of host insects to facilitate Metarhizium infection

Insects have an effective innate immune system to protect themselves against fungal invasion. Metarhizium employs a toxin-based strategy using a nonribosomal peptide called destruxin A (DA) to counteract the host immune response. However, the mechanism by which DA inhibits insect immunity is still unclear. Here, we identified 48 DA-binding proteins in silkworm hemolymph, with the binding affinity (KD) ranging from 2 to 420μM. Among these proteins, hemocytin, an important immune factor, was determined to be the strongest DA-binding protein. DA binds to hemocytin and regulates its conformation in a multisite manner. Furthermore, DA exerts a significant inhibitory effect on hemocytin-mediated hemocyte aggregation. By disrupting the interaction between hemocytin, actin A3, and gelsolin, DA prevents the transformation of granules into vesicles in hemocytes. These vesicles are responsible for storing, maturing, and exocytosing hemocytin. Therefore, hemocytin secretion is reduced, and the formation of structures that promote aggregation in outer hemocytes is inhibited.