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Eicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450-derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

BACKGROUND: Owing to its systemic mode‐of‐action and ease of application, lithium chloride (LiCl) is an ideal varroacide for the control of Varroa destructor infestations in honey bee colonies. To better understand how LiCl functions within a colony, we screened different parts of honey bee anatomy for lithium accumulation. We wanted to elucidate the time‐dependent effects of LiCl on V. destructor and its metabolism within honey bees when they were fed continuous LiCl treatments, as well as evaluate potential adverse effects such as accumulation in the hypopharyngeal glands of nurse bees, which could negatively impact queens and larvae. RESULTS: Cage experiments reveal rapid acaricidal onset, with >95% mite mortality within 48 h of treatment. Bee hemolymph analysis supports these observations, showing a rapid increase in lithium concentration within 12 h of treatment, followed by stabilization at a constant level. Lithium accumulates in the rectum of caged bees (≤475.5 mg kg−1 after 7 days of feeding 50 mm LiCl), reflecting the bees' metabolic and excretion process. Despite concerns about potential accumulation in hypopharyngeal glands, low lithium levels of only 0.52 mg kg−1 suggest minimal risk to the queen and 1st‐ and 2nd‐instar larvae. Cessation of LiCl treatment results in a rapid decline in mite mortality in the first 5 days, which increases again thereafter, resulting in mite mortality of 77–90% after 10 days. CONCLUSION: These findings help optimize LiCl application in colonies to achieve high Varroa mortality without unwanted adverse effects and provide important baseline data for future registration. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Development of multidrug resistance in infectious microbes is a common phenomenon and hence the search for alternatives with better activity continues. Silkworm antimicrobial peptides have been quite resourceful in this aspect as they are known to be potent against various bacteria, viruses and fungi. This study assesses the antimicrobial potentials of the peptides isolated from Bacillus thuringiensis immunised 5th instar lava of the Muga silkworm (Antheraea assamensis). The proteins produced as a result of immunization were isolated from the haemolymph and purified partially by acid-methanolic extraction which was then subjected for separation and purification by semi preparative RP-HPLC. The collected fractions were analysed for purity by SDS-PAGE following which, antimicrobial activity assay was performed against Gram-positive (Bacillus thuringiensis) and Gram-negative bacteria (Escherichia coli). Among the obtained eight purified fractions, seven fractions showed different ranges of antimicrobial activity. Amongst them, two fractions were seen to show antimicrobial activity against both Gram positive as well as Gram negative bacteria. [ABSTRACT FROM AUTHOR]

Hemocytes are pivotal in the immune response of insects against invasive pathogens. However, our knowledge of hemocyte types and their specific function in Tribolium castaneum, an increasingly important Coleoptera model insect in various research fields, remains limited. Presently, a combination of morphological criteria and dye‐staining properties were used to characterize hemocyte types from T. castaneum larvae, and 4 distinct types were identified: granulocytes, oenocytoids, plasmatocytes and prohemocytes. Following different immune challenges, the total hemocyte counts declined rapidly in the initial phase (at 2 h), then increased over time (at 4 and 6 h) and eventually returned to the naive state by 24 h post‐injection. Notably, the morphology of granulocytes underwent dramatic changes, characterized by an expansion of the surface area and an increased production of pseudopods, and with the number of granulocytes rising significantly through mitotic division. Granulocytes and plasmatocytes, the main hemocyte types in T. castaneum larvae, can phagocytose bacteria or latex beads injected into the larval hemolymph in vivo. Furthermore, these hemocytes participate in the encapsulation and melanization processes in vitro, forming capsules to encapsulate and melanize nickel–nitrilotriacetic acid (Ni–NTA) beads. This study provides the first comprehensive characterization of circulating hemocytes in T. castaneum larvae, offering valuable insights into cell‐mediated immunity in response to bacterial infection and the injection of latex beads. These results deepen our understanding of the cellular response mechanisms in T. castaneum larvae and lay a solid foundation for subsequent investigations of the involvement of T. castaneum hemocytes in combating pathogens. [ABSTRACT FROM AUTHOR]

Since 2000, a well‐established population of the invasive oriental shrimp Palaemon macrodactylus has been present in fully marine conditions in the southwestern Atlantic Ocean (~38° S). To assess the physiological performance of this atypical population restricted to fully marine conditions, we conducted a laboratory experiment in which individuals were transferred from 35 ‰S (local seawater) to 2 ‰S; 5 ‰S; 10 ‰S; 20 ‰S; 50 ‰S and 60‰ for short (6 h), medium (48 h), and long (>504 h) acclimation periods. We measured the time course response of relevant parameters in the shrimp's hemolymph; activity of Na+, K+‐ATPase (NKA), and V‐H+‐ATPase (VHA); and muscle water content. Shrimp showed great osmoregulatory plasticity, being able to survive for long periods between 5 ‰S and 50 ‰S, whereas no individual survived after transfer to either 2 ‰S or 60 ‰S. Shrimp hyper‐regulated hemolymph osmolality at 5 ‰S and 10 ‰S, hypo‐regulated at 35 ‰S and 50 ‰S, and isosmoticity was close to 20 ‰S. Compared to 35 ‰S, prolonged acclimation to 5 ‰S caused a decrease in hemolymph osmolality (~34%) along with sodium and chloride concentrations (~24%); the NKA and VHA activities decreased by ~52% and ~88%, respectively, while muscle water content was tightly regulated. Our results showed that the atypical population of P. macrodactylus studied here lives in a chronic hypo‐osmo‐ion regulatory state and suggest that fully marine conditions contribute to its poor performance at the lower limit of salinity tolerance (<5 ‰S). Research Highlights: Shrimp lives in a chronic hypo‐osmo‐ionic regulatory state.Shrimp can tolerate brackish water but not freshwater.An incipient local physiological adaptation to full‐strength seawater is suggested. [ABSTRACT FROM AUTHOR]

The issue of microplastics (MPs) has emerged as a significant concern globally, with discussions surrounding the potential environmental impact of these tiny plastic particles becoming increasingly prevalent. This study aimed to identify the concentration and characteristics of MPs in hemolymph and organs (gills and hepatopancreas) of green mussels (Perna viridis) that are frequently consumed by people in Pangkajene Kepulauan, South Sulawesi Province, Indonesia. Green mussels were collected from two different sampling sites for comparison. Screening was carried out on dispensed hemolymph and dissected organs to identify the characteristics of MPs. Surface seawater sampling was added as information on MP's characteristics from the mussel habitat. Visual observation of MP's characteristics using a stereomicroscope in laminar flow is to prevent contamination. The identification of MP's polymer type is using FTIR-ATR. The results showed that hemolymph, hepatopancreas, gills, and surface water were concentrated with MPs. Small (2–3.9 cm) green mussels accumulated more MPs than medium (4–5.9 cm) and large (> 6 cm). MPs characteristics of fiber shape, transparent color, and size 0.1–0.5 mm were dominant in all samples. A total of seven polymers of MPs were identified with polyethylene and polystyrene types most frequently found from all samples. Based on this study, green mussels are good for biomonitoring of MPs. [ABSTRACT FROM AUTHOR]

Nutrition is vital to health, but while the link between diet and body nutritional composition is well explored in humans and other vertebrates, this information is not well understood in insects, despite the vital roles they play in ecosystems, and their increasing use as experimental models. Here we used Nutritional Geometry to explore the rapid physiological response to ingested nutrients in the haemolymph nutritional profile of Spodoptera littoralis caterpillars. We ask whether blood nutrients are maintained homeostatically in the face of variable nutritional intake, or if regulation is more flexible for some nutrients than others (allostasis), which allows animals to adapt to stress by responding in a way that prioritises efficiency of responses in the face of trade‐offs. Caterpillars were placed on 1 of 20 diets, systematically varying in their nutrient ratios (protein: carbohydrate) and density (calorie content), and their consumption was measured. After 48 h, caterpillars were bled, and the macronutrient (protein, carbohydrates and lipids) and nutrient metabolite (amino acids and simple sugars) content of the haemolymph was measured. Proteins comprised 93% of the haemolymph macronutrient pool on average and their concentration increased with protein eaten. The amino acid (AA) pool was dominated by five AAs, and the total pool increased with total nutrient intake. However, the ratio of essential to non‐essential AAs increased as the proportion of protein consumed increased. Carbohydrates were tightly controlled, increasing only on the most extreme carbohydrate intakes. Simple sugars were dominated by glucose and trehalose, and overall, the simple sugar pool showed high levels of homeostasis. Rather than strict homeostasis of blood nutritional properties, an allostatic model seemed to be a better fit for blood nutrient regulation in this generalist herbivore. This flexibility in response to the nutritional composition of the diet may, in part, explain how this species has evolved to extreme dietary generalism and may play a role in its worldwide pest status. Given the range of fitness‐related processes affected by the haemolymph, future studies should examine the physiological impacts of blood nutrient variation on reproduction, growth and response to infection and the trade‐offs between them. [ABSTRACT FROM AUTHOR]

The leaf‐cutter ant Atta sexdens is crucial in ecological balance and economic activities, due to its contribution to the aeration and accumulation of organic matter in the soil, but it may also be an agricultural pest when it defoliates plants for the fungus growth used as a food source. This leaf‐cutter ant exhibits a polymorphic worker caste system with distinct activities in the colony. These functions are performed by workers classified into subcastes, including gardeners, which are responsible for the fungus and brood care; cleaners (waste removers), which remove wastes; foragers, which collect and transport plant parts to the nest; and soldiers, which are responsible for colony defence and the support of foraging activities. In our previous work, we showed that A. sexdens has five types of circulating haemocytes in the haemolymph, which play a vital role in immune defence. Herein, we further investigated the population profile of circulating haemocytes in the haemolymph of different worker subcastes of A. sexdens using flow cytometry. Each subcaste exhibited a single population of haemocytes in the haemolymph. However, a detailed 3D dispersion analysis uncovered five distinct haemocyte subpopulations, two of which presented higher quantities. The soldier displayed haemocytes with high fluorescence intensity, compared with the gardener, waste remover, and forager, along with increased complexity, compared with the gardener subcaste. These results suggest that these cells may be granulocytes and plasmatocytes, which are known for their role in insect immune defence. [ABSTRACT FROM AUTHOR]

Abstract Background The acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be interesting models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., ‘uncovered lungs’ in Nagurus okinawaensis (Trachelipodidae), ‘dorsal respiratory fields’ in Alloniscus balssi (Alloniscidae), and pleopods without respiratory structures in Armadilloniscus cf. ellipticus (Detonidae). Results In N. okinawaensis with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in Al. balssi with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in Ar. cf. ellipticus, only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis. Conclusions This study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the epithelial positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields. This suggests that these two types of respiratory structures do not result from simple differences in the degree of development. Future analysis of molecular developmental mechanisms will help determine whether these are the result of heterotopic changes or have different evolutionary origins. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs.

Sawfly species of the genus Monophadnus are specialised on Ranunculaceae plants from which the larvae can sequester furostanol saponins into the haemolymph, mainly (25R)-26-[(α-L-rhamnopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl-O-β-D-glucopyranosyl-(1→3)-O-[6-acetyl-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranoside (compound 1). In this work, TLC, GC-MS, and HPLC-DAD-ESI/MS analyses together with feeding, repeated simulated attacks, and ant deterrence bioassays were conducted to extend the chemoecological knowledge about two sawfly species specialised on H. foetidus L. (Monophadnus species A) and H. viridis L. (Monophadnus species B). Larvae of Monophadnus species B were mostly feeding on the squares treated with the n-butanol fraction from H. foetidus, compound 1 being its primary non-nutritional stimulant. In contrast, all H. viridis fractions stimulated feeding, with n-hexane marginally more active. β-sitosterol within n-hexane was determined as the nutritional stimulant. Quantitative analyses demonstrated that leaves of H. viridis but not H. foetidus contain the ecdysteroids 20-hydroxyecdysone and polypodine B. Moreover, the haemolymph of Monophadnus species B larvae reared on H. viridis contained the glycosides of polypodine B and 20-hydroxyecdysone at a concentration of 2.5 to 6.8 µmol/g fresh weight of haemolymph. This concentration is several thousand times higher than the concentration range of the aglycones in their host plant (3.63 × 10−4 to 2.23 × 10−4 µmol total ecdysteroids/g fresh weight of leaves), suggesting bioaccumulation. The larvae of both species fed on H. foetidus do not show any traces of ecdysteroids in their haemolymph, indicating a facultative role of these compounds in their defence as well as their inability to endogenously synthesise these compounds. The haemolymph containing ecdysteroids was a significant feeding deterrent against Myrmica rubra L. ant workers (one of their natural predators) at 0.8 mg/mL. The larvae kept effective deterrent levels of glycosylated ecdysteroids (≅175 mM) between simulated attacks on days 1 and 2, but the levels clearly decreased on day 3 (≅75 mM). Most larvae (89%) survived a first attack but only 23% a consecutive second one. As a conclusion, we report for the first time that two Monophadnus species feeding on H. viridis sequester phytoecdysteroids into the larval haemolymph in the form of glycosides. In addition, compound 1 possesses defensive and phagostimulant activities, and we present evidence for a combined effect of furostanol saponins and ecdysteroids as repellents against ants. [ABSTRACT FROM AUTHOR]

【Objective】We investigated the changes of GlcNAcases activity in different tissues of the fifth instar of Bombyx mori after treatment with 20-hydroxyecdysone (20E) and Juvenile hormone (JH) respectively, aiming to provide references for the further analysis of the biological function of β-N-acetylgucosaminidase (GlcNAcases）.【Method】The changes of GlcNAcases activity in hemolymph, fatbody, midgut, integument and silkgland of silkworm larvae were measured and analyzed by spectrophotometry at 6, 12, 18, 24 and 48 h after injecting 20E and JH with microinjector.【Result】In the JH treatment group, the GlcNAcases activity began to increase significantly at 12 h and continued to be significantly higher than that in the control group at 18, 24 and 48 h in hemolymph. However, after 20E treatment, the GlcNAcases activity was significantly higher than that in the control group only at 24 and 48 h. After 20E or JH treatment, the changes of GlcNAcases activity was similar in fatbody. There was no significant change at 6, 12 and 18 h, but the GlcNAcases activity was significantly higher than that of the control group at 24 and 48 h. In the midgut, the GlcNAcases activity was slightly higher than that of the control group at 6 and 12 h after 20E or JH treatment, but the difference was not significant compared with the control group. However, the GlcNAcases activity was significantly higher than that of the control group at 18 h and continued to increase at 24 h. After that, it began to decrease to the level of the control group at 48 h. In the integument, the GlcNAcases activity was significantly higher than that of the control group at 12 h and continued to be significantly higher at 18 and 24 h, but it decreased to the level of the control group at 48 h after 20E treatment. While in JH treatment group, the GlcNAcases activity was significantly lower than that of the control group at 12, 18 and 24 h and recovered to the level of the control group at 48 h. In the silkgland, the GlcNAcases was higher than that of the control group at 12 h after 20E treatment, but the difference was not significant. Thereafter, the enzyme activity continued to increase and were significantly higher than that of the control group at 18 and 24 h. At 48 h, the enzyme activity decreased to a certain extent, but was still significantly higher than that of the control group. In the JH treatment group, the enzyme activity began to decrease from 12 to 48 h and the difference reached a significant or extremely significant level compared with the control group at 18, 24 and 48 h.【Conclusion】20E and JH could activate GlcNAcases activity in the hemolymph, fatbody and midgut of silkworm larvae. However, in the integument and silk gland, the GlcNAcases were activated by 20E but inhibited by JH. The results indicated that GlcNAcases plays diverse functions in different tissues of Bombyx mori and the effects of different hormones on the GlcNAcases activity were also distinct. [ABSTRACT FROM AUTHOR]

Microplastic (MP) contamination in the aquatic environment is a cause of concern worldwide since MP can be taken up by different organisms, altering different biological functions. In particular, evidence is accumulating that MP can affect the relationship between the host and its associated microbial communities (the microbiome), with potentially negative health consequences. Synthetic microfibers (MFs) represent one of the main MPs in the marine environment, which can be accumulated by filter-feeding invertebrates, such as bivalves, with consequent negative effects and transfer through the food chain. In the mussel Mytilus galloprovincialis, polyethylene terephthalate (PET) MFs, with a size distribution resembling that of an MF released from textile washing, have been previously shown to induce multiple stress responses. In this work, in the same experimental conditions, the effects of exposure to PET-MF (96 h, 10, and 100 μg/L) on mussel hemolymph microbiome were evaluated by 16S rRNA gene amplification and sequencing. The results show that PET-MF affects the composition of bacterial communities at the phylum, family and genus level, with stronger effects at the lowest concentration tested. The relationship between MF-induced changes in hemolymph microbial communities and responses observed at the whole organism level are discussed. [ABSTRACT FROM AUTHOR]

To better cope with unfavorable climatic conditions and perform optimal physiological functions, the silkworm Bombyx mori secretes some of its defensive enzymes in the hemolymph. To understand the extent of enzyme secretion in the hemolymph, we studied the profiles of defense enzymes in different silkworm breeds. We collected and analyzed the hemolymph of 5th instar larvae of 21 different bivoltine silkworm breeds with different characteristics for protein content and activities of enzymes such as catalase, peroxidase, polyphenol oxidase, amylase, and protease. The results revealed significant differences between the breeds in terms of protein and defensive enzyme activities. The activity of catalase, peroxidase, polyphenol oxidase, amylase, and protease varied between silkworm breeds, with an average activity of 8.791 U mg−1, 0.203 U mg−1, 0.42 U mg−1, 0.264 mg ml−1 min−1 mg−1 and 0.193 µg ml−1 min−1, respectively. To understand the genetic relationship between the silkworm breeds in terms of the activity of defensive enzymes, we constructed a dendrogram using Euclidean distance. The 21 silkworm breeds were grouped into five different clusters with different enzyme activities, revealing sufficient diversity between the silkworm breeds that can be used for parental selection in silkworm breeding programs. This study is highly informative in enhancing knowledge on the role of defensive enzymes in the development of new silkworm breeds that can adapt to adverse environmental conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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). Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Background: The acquisition of air-breathing organs is one of the key innovations for terrestrialization in animals. Terrestrial isopods, a crustacean lineage, can be interesting models to study the evolution of respiratory organs, as they exhibit varieties of air-breathing structures according to their habitats. However, the evolutionary processes and origins of these structures are unclear, due to the lack of information about their developmental processes. To understand the developmental mechanisms, we compared the developmental processes forming different respiratory structures in three isopod species, i.e., 'uncovered lungs' in Nagurus okinawaensis (Trachelipodidae), 'dorsal respiratory fields' in Alloniscus balssi (Alloniscidae), and pleopods without respiratory structures in Armadilloniscus cf. ellipticus (Detonidae). Results: In N. okinawaensis with uncovered lungs, epithelium and cuticle around the proximal hemolymph sinus developed into respiratory structures at post-manca juvenile stages. On the other hand, in Al. balssi with dorsal respiratory fields, the region for the future respiratory structure was already present at manca 1 stage, immediately after hatching, where the lateral protrusion of ventral epithelium occurred, forming the respiratory structure. Furthermore, on pleopods in Ar. cf. ellipticus, only thickened dorsal cuticle and the proximal hemolymph sinus developed during postembryonic development without special morphogenesis. Conclusions: This study shows that the respiratory structures in terrestrial isopods develop primarily by postembryonic epithelial modifications, but the epithelial positions developing into respiratory structures differ between uncovered lungs and dorsal respiratory fields. This suggests that these two types of respiratory structures do not result from simple differences in the degree of development. Future analysis of molecular developmental mechanisms will help determine whether these are the result of heterotopic changes or have different evolutionary origins. Overall, this study provides fundamental information for evolutionary developmental studies of isopod respiratory organs. [ABSTRACT FROM AUTHOR]

Human activities associated with large-scale farms and the monocultures expose honey bees to one type of food. Moreover, there is an ongoing decline of plant species producing pollen and nectar in Europe. A poorly balanced diet affects a number of processes occurring in a bee's body. The fat body and hemolymph are the tissues that participate in all of them. Therefore, the aim of our study was to determine the effect of hazel, pine, rapeseed, buckwheat, phacelia and goldenrod pollen on the morphological parameters of fat body trophocytes, the diameters of cell nuclei in oenocytes and the concentrations of compounds involved in energy metabolism (glucose, glycogen, triglycerides and protein). In the cage tests, the bees were fed from the first day of life with sugar candy (control group) or candy with a 10% addition of one of the 6 pollen types. Hemolymph and fat body from various locations were collected from 1-, 7- and 14-day-old workers. Pollen produced by plant species such as hazel and pine increased glucose concentrations in the bee tissues, especially in the hemolymph. It can therefore be concluded that they are valuable sources of energy (in the form of simple carbohydrates) which are quickly used by bees. Pollen from plants blooming in the summer and autumn increased the concentrations of proteins, glycogen and triglycerides in the fat body, especially that from the third tergite. The accumulation of these compounds was associated with an increased the length and width of trophocytes as well as with enhanced metabolic activity, which was evidenced in the increasing diameter of oenocyte cell nuclei. It seems a balanced multi-pollen diet is more valuable for bees, but it is important to understand the effects of the particular pollen types in the context of a mono-diet. In the future, this will make it possible to produce mixtures that can ensure homeostasis in the apian body. [ABSTRACT FROM AUTHOR]

Mytilus is one of bivalves with great economic and ecological values. The innate immune defense of Mytilus shows great significance in the study of marine biological immunology. Hemolymph is the main immune tissue for Mytilus. The Nanopore full-length transcriptome of Mytilus coruscus hemocytes, and the serum differential proteomics based on SDS-PAGE analysis were performed to identify key proteins involving in the immune response of Mytilus hemolymph in response of different bacteria and fungi stresses. A total of 44 proteins were identified in the serum induced by different microorganisms. Among them, 26 proteins showed significant differential expression level in response to different microbial stresses, and their functions were involved in protein folding protection, cell autophagy and apoptosis regulation, reactive oxygen species production, energy metabolism regulation, cell detoxification, and immune regulation. The changes in expression levels of these proteins varied in response to different bacterial and fungal stresses, suggesting that Mytilus has different immune response strategies to different bacterial and fungal stresses. The results provide a new scientific basis for understanding the differential immune mechanism of Mytilus innate immune system in response to different types of microbial invasion, as well as the screening of specific biomarker proteins for microbial infection, and provide ideas for the healthy development and disease prevention of shellfish aquaculture. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bivalves play a key role in aquatic ecosystems and are a valuable commercial resource. The prosperity of these aquatic organisms depends mainly on the effectiveness of their immune defense, in which the hemolymph plays a central role. Hemolymph may be used as an effective non-lethal criterion of health. However, the predictive value of hemolymph analysis depends on the comparison between the obtained results and reference data from healthy individuals living in natural aquatic environments. We collected hemolymph from 15 commercially important species from wild populations at stations located in non-impacted and impacted water areas of the Sea of Japan. Of the 11 hemolymph parameters we analyzed, the total hemocyte count, percentage of hemocyte types, phagocytic activity, presence of reactive oxygen species, and protein concentration differed significantly between populations from non-impacted and impacted water areas. The most responsive species to pollution were Magallana gigas, Crenomytilus grayanus, Mizuhopecten yessoensis, and Mactra chinensis. This work is the first to examine a large number of commercially important species simultaneously. The results of this study are the basis for establishing the health status criteria of commercial bivalves for veterinary control in aquaculture and biomonitoring. [ABSTRACT FROM AUTHOR]

The Pacific oyster Crassostrea gigas, one of the most exploited molluscs in the world, has suffered from massive mortality in recent decades, and the occurrence mechanisms have not been well-characterized. In this study, to reveal the relationship of associated microbiota to the fitness of oysters, temporal dynamics of microbiota in the gill, hemolymph, and hepatopancreas of C. gigas during April 2018–January 2019 were investigated by 16S rRNA gene sequencing. The microbiota in C. gigas exhibited tissue heterogeneity, of which Spirochaetaceae was dominant in the gill and hemolymph while Mycoplasmataceae enriched in the hepatopancreas. Co-occurrence network demonstrated that the gill microbiota exhibited higher inter-taxon connectivity while the hemolymph microbiota had more modules. The richness (Chao1 index) and diversity (Shannon index) of microbial community in each tissue showed no significant seasonal variations, except for the hepatopancreas having a higher richness in the autumn. Similarly, beta diversity analysis indicated a relatively stable microbiota in each tissue during the sampling period, showing relative abundance of the dominant taxa exhibiting temporal dynamics. Results indicate that the microbial community in C. gigas showed a tissue-specific stability with temporal dynamics in the composition, which might be essential for the tissue functioning and environmental adaption in oysters. This work provides a baseline microbiota in C. gigas and is helpful for the understanding of host-microbiota interaction in oysters. [ABSTRACT FROM AUTHOR]

In recent years, there has been a growing interest in exploring novel natural sources for antioxidant and anti-inflammatory agents, driven by the need for alternative therapeutic strategies with fewer side effects. This study investigates the potential of the Biomphalaria alexandrina snail as a valuable model organism to evaluate the antioxidant and anti-inflammatory properties of Sarcophaga argyrostoma larval hemolymph. The choice of B. alexandrina snails as a model is based on its ecological relevance and physiological similarities to other organisms, supporting well-established use in ecological and physiological research, as well as it provides a valuable platform for such investigations. Ultimately, this study may open avenues for the development of innovative and sustainable therapeutic interventions rooted in the rich biodiversity of the natural world. The experimental design involves the extraction of hemolymph from S. argyrostoma larvae, followed by a comprehensive analysis of its antioxidant capacity and anti-inflammatory effects using adult snails. Various biochemical assays, including total antioxidant capacity, immunomodulatory markers and histological investigation, will be employed to assess the bioactive potential of the hemolymph. The outcomes of this research can be attributed to the identification of the anti-oxidant and anti-inflammatory effects of hemolymph. This effect was observed through the reduction of oxidative stress and proinflammatory mediators, impacting the biological activities of the snails. The study is the first to shed light on the potential benefits of S. argyrostoma larval hemolymph using an animal model, showcasing its ability to mitigate oxidative and inflammatory responses within the biological context of the snails. [ABSTRACT FROM AUTHOR]

The sex of Lepidoptera species can typically be identified at the pupal or adult stage by their morphological characteristics. However, for most species, no obvious sex‐specific traits can be used at the larval or embryonic stage. The growing interest in studying sex determination and differentiation, along with the potential application of research findings in genetic regulation techniques, is promoting the advancement of new sexing methods at early developmental stages. In this study, the sex of individual eggs and larvae was successfully identified by means of W chromosome molecular markers, which were identified through analysing the previously published W chromosome sequence data of Spodoptera frugiperda (Lepidoptera: Noctuidae). Additionally, we present methods to extract DNA and RNA from individual eggs and larval hemolymph. These techniques provide a simple and dependable method to identify sex at pre‐pupal stages. This approach could potentially be extended to other Lepidoptera species of which the W chromosome information is available. [ABSTRACT FROM AUTHOR]

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Dinoflagellates belonging to the genus Hematodinium are key parasites of marine crustaceans, primarily decapods. In this study, we document the first report of H. perezi Chatton & Poisson, 1930 on the African Atlantic coast. This is also the first parasite record in the invasive non-native Atlantic blue crab Callinectes sapidus Rathbun, 1896 in Morocco. Specimens of C. sapidus were sampled in winter 2023 from two Ramsar sites on the Moroccan Atlantic, namely Merja Zerga and Oualidia Lagoons, and were screened to detect the presence of parasites in their hemolymph. Based on staining fresh hemolymph smears, we did not detect Hematodinium in any of the 36 investigated individuals (20 and 16 from Merja Zerga and Oualidia Lagoons, respectively), probably due to methodological artifacts. The PCR-based method was revealed to be more accurate in diagnosing the Hematodinium parasite. It showed that at Merja Zerga Lagoon, 13 individuals of C. sapidus were infected by the parasite (prevalence: 65%) in comparison to four at Oualidia Lagoon (25%). Genetic analysis, based on the ITS1 rDNA gene from Hematodinium, confirmed the sequences as being those of Hematodinium perezi. [ABSTRACT FROM AUTHOR]

Multiple drug-resistant fungal species are associated with the development of diseases. Thus, more efficient drugs for the treatment of these aetiological agents are needed. Rondonin is a peptide isolated from the haemolymph of the spider Acanthoscurria rondoniae. Previous studies have shown that this peptide has antifungal activity against Candida sp. and Trichosporon sp. strains, acting on their genetic material. However, the molecular targets involved in its biological activity have not yet been described. Bioinformatics tools were used to determine the possible targets involved in the biological activity of Rondonin. The PharmMapper server was used to search for microorganismal targets of Rondonin. The PatchDock server was used to perform the molecular docking. UCSF Chimera software was used to evaluate these intermolecular interactions. In addition, the I-TASSER server was used to predict the target ligand sites. Then, these predictions were contrasted with the sites previously described in the literature. Molecular dynamics simulations were conducted for two promising complexes identified from the docking analysis. Rondonin demonstrated consistency with the ligand sites of the following targets: outer membrane proteins F (id: 1MPF) and A (id: 1QJP), which are responsible for facilitating the passage of small molecules through the plasma membrane; the subunit of the flavoprotein fumarate reductase (id: 1D4E), which is involved in the metabolism of nitrogenous bases; and the ATP-dependent Holliday DNA helicase junction (id: 1IN4), which is associated with histone proteins that package genetic material. Additionally, the molecular dynamics results indicated the stability of the interaction of Rondonin with 1MPF and 1IN4 during a 10 ns simulation. These interactions corroborate with previous in vitro studies on Rondonin, which acts on fungal genetic material without causing plasma membrane rupture. Therefore, the bioprospecting methods used in this research were considered satisfactory since they were consistent with previous results obtained via in vitro experimentation. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGNdegrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The current study investigated proteomic divergence in the hemolymph of early fourth instar larvae of Helicoverpa armigera fed with triflumuron (TFM) and a synergistic growth inhibitory combination of TFM and β-sitosterol (BS) in a 1:1 ratio. LC-MS analysis identified 65 proteins differentially downregulated in larvae fed with TFM and 75 in larvae fed with TFM + BS, essential for maintaining homeostasis and performing physiological and metabolic activities in H. armigera, when compared to untreated controls. In the TFM-fed larvae, deletions comprised 7 DNA processing, 11 RNA processing, 7 protein processing, 6 metabolic, 5 transport, 11 structural, 8 signal transduction, and 10 stress response proteins. TFM + BS-fed larvae showed relative deletions of 8 DNA processing, 14 RNA processing, 6 protein processing, 7 metabolic, 7 transport, 13 structural proteins, 7 signal transduction, and 12 stress response proteins. Both diets resulted in differential expression of 10 and 6 proteins, respectively, with TFM-fed larvae expressing proteins related to defense and detoxification, while TFM + BS-fed larvae expressing primarily upregulated proteins involved in sensory stimulus perception, particularly olfaction, along with one stress protein. Thus, analysis of the overall protein expression of the organism could provide an insight into important proteins suppressed and activated which can be future targets protein to control the pest population. [ABSTRACT FROM AUTHOR]

Urban beekeeping has gained interest in recent years. Bees placed in cities, on the roofs of buildings, are exposed to many different sources of electromagnetic fields of different frequencies and intensities. Knowledge about the impact of electromagnetic fields on the physiology and behavior of insects (including honey bees) is limited. Hence, one of the first steps was to check how radiofrequency electromagnetic fields affect honey bees' nutrition. The level of total proteins, glucose, triglycerides, and TAS activity in bee hemolymph was analyzed. This is because they indicate the honey bee's nutrition level. The experiment involved 2-day-old Carniolan worker honey bees (Apis mellifera carnica L.). The bees were exposed to fields of 12 V/m, 28 V/m, and 61 V/m for 0.25, 1 and 3 h. Hemolymph was taken from 40 alive worker honey bees from each group immediately after exposure, by removing the antennae with sterile tweezers. The activity of biochemical markers (total proteins, TAS, and triglycerides) in the bee hemolymph in 12 V/m and 28 V/m groups showed statistically significant differences compared to the control group in most cases. According to our own research, radiofrequency electromagnetic fields disturb honey bee nutrition. The changes in the analyzed indicators may have long-term effects. [ABSTRACT FROM AUTHOR]

Simple Summary: Currently, the optimization of mass rearing conditions for insects is a matter of highly applied and fundamental relevance since insects can be used as a source of protein in feed and feed supplements for farm animals as well as a source of antibacterial compounds for drug development and various insect research activities in laboratory conditions. The quantity and quality of the consumed feed has an effect on insect growth rate, duration of development, body weight, ability to spread, and survival, as well as on the mating success rate and total egg production of females. Suboptimal nutrition, growth, and development can affect beetle viability. We developed an artificial fungi-based diet for rearing an insect group adapted to life in wood and using it as one of their food sources and tested the effect of this diet on various parameters of the insects' development and their immunity. Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) was chosen as a model species (representative of this group of insects). Based on the parameters of development at all stages of Z. atratus and screening of hemolymph antibacterial activity of their larvae, we showed that the cultivation method we developed shortens the development time of beetles and strengthens their immunity. The rearing of saproxylic insects in laboratory conditions is an important task for studying the biology of insects. Through understanding nutritional needs, it is possible to optimize beetle rearing in laboratory conditions. In this study, an artificial fungi-based diet (FD) was developed for the cultivation of the darkling beetle Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) in laboratory conditions as a model object for studying the biology of saproxylophagous beetles. To assess the influence of the diet, a number of physiological parameters were measured, including development time, body size, and weight of all stages of the beetle's life cycle, as well as its immune status. The immune status of Z. atratus was assessed on the basis of larval hemolymph antibacterial activity against six different bacterial strains assessed using disk-diffusion and photometric tests. Our findings show that the FD reduces development time and boosts the immune status as compared to beetles reared on a standard diet (SD). Samples from FD-reared larvae had pronounced antibacterial activity as compared to samples from SD-reared larvae. This work is of fundamental importance for understanding the correlations between nutrition and development of saproxylic Coleoptera and is the first report on immune status regulation in this group of insects. [ABSTRACT FROM AUTHOR]

Hemocytes are specialized hemolymph cells found in insects, responsible for a variety of important physiological functions such as immunity, wound healing, and the regulation of the insect's circulatory system. The study of insect hemocytes has become increasingly important in recent years due to the emergence of insect-borne diseases and the need to develop new strategies for insect control. Understanding the biology of insect hemocytes has the potential to lead to the development of novel insecticides and strategies for controlling insect-borne diseases. This study aimed to examine the hemocytes in Bombyx mori to determine if there were any variations in the types of hemocytes to other arthropods. By comprehensively characterizing hemocyte types across developmental stages, this research sheds light on the intricate immune responses and adaptations of the silkworm. Such insights could contribute to the understanding of basic immunological mechanisms in insects. Hemolymph of B. mori was collected, and hemolymph smears were stained with Wright’s stain. In B. mori, four distinct types of hemocyte prohemocyte, plasmatocyte, granulocyte, and oenocytoid were examined. Using light microscopy, the hemocyte types were identified based on their size, the presence or absence of granules, and the ratio of their nucleus to cytoplasm. [ABSTRACT FROM AUTHOR]

Combining microbial products with different mechanisms of action can produce synergistic insecticidal effects and slow down the development of resistance. This study evaluated the insecticidal activity of mixtures containing the commercial Photorhabdus luminescens (Pl) strain ATCC 29,999, the local isolate Pl 2103-UV, and the commercial Bacillus thuringiensis subsp. kurstaki (Bt) ABTS-351 against Spodoptera frugiperda. As the proportion of Bt increased in the mixtures, insecticidal activity increased, with the synergistic ratios reaching 1.98 for ATCC 29,999 and 5.29 for 2103-UV at a1:5 Pl:Bt ratio, representing approximately twofold and fivefold reductions, respectively, in the Bt dosage required for mortality. Hemolymph analysis revealed the highest Pl bacterial loads in the 1:5 treatments within the S. frugiperda hemocoel. Histopathology also showed exacerbated midgut vacuolation in the 1:5 ATCC 29,999:Bt treatment. Whole-genome analysis showed that 2103-UV produced more porins, potentially contributing to its higher insecticidal activity. This study demonstrated that Bt assists the invasion of Pl into the hemocoel and enhances synergistic insecticidal efficiency. The findings provide a reference for integrating Pl with other microbial products to sustainably manage significant and severe lepidopteran pests. [ABSTRACT FROM AUTHOR]

Abstract: Eicosanoids are a class of molecules derived from C20 polyunsaturated fatty acids (PUFAs) that play a vital role in mammalian and insect biological systems, including development, reproduction, and immunity. Recent research has shown that insects have significant but lower levels of C20 PUFAs in circulation in comparison to C18 PUFAs. It has been previously hypothesized in insects that eicosanoids are synthesized from C18 precursors, such as linoleic acid (LA), to produce downstream eicosanoids. In this study, we show that introduction of arachidonic acid (AA) stimulates production of cyclooxygenase, lipoxygenase, and cytochrome P450–derived eicosanoids. Downstream immune readouts showed that LA stimulates phagocytosis by hemocytes, while both LA and AA stimulate increased antimicrobial peptide production when D. melanogaster is exposed to a heat-killed bacterial pathogen. In totality, this work identifies PUFAs that are involved in insect immunity and adds evidence to the notion that Drosophila utilizes immunostimulatory lipid signaling to mitigate bacterial infections. Our understanding of immune signaling in the fly and its analogies to mammalian systems will increase the power and value of Drosophila as a model organism in immune studies.

Aim. The concentration of total protein in the hemolymph of the Australian red-clawed crayfish Cherax quadricarinatus, Von Martens 1868, contained in artificial conditions in closed water supply installations and an aquarium complex, was studied. Methodology. The object of the study was the Australian red-clawed crayfish, a total of 161 individuals were used in the experiment. Crayfish were kept in closed water supply installations. To study the protein concentration depending on the time of year, samples were taken and analyzed in the period 2021-2023: January – winter; April – spring; August – summer; September, October and November – autumn. To conduct an experiment to study the total protein content of cancer hemolymph, depending on the water temperature, three experimental groups of 15 cancers were formed in each. The experimental groups were seated in three aquariums, with a water temperature of 20, 25, 30 ° C. The duration of the crayfish exposure was 10 days. On the tenth day, hemolymph was collected. The total hemolymph protein was determined by the refractometric method. Results. It was found that the concentration of total protein in the hemolymph can be in the ranges from 10.8 to 108.8 g/l and average 50.5 ± 21.1 g/l. In crustaceans kept in unfavorable conditions, the concentration of total protein was 1.6 times lower. There were no differences in protein concentration at different seasons of the year. After molting, the concentration of total protein in the hemolymph decreases 2.7 times. It was found that short-term exposure of cancer in water with a temperature of 20, 25, 30 ° C does not lead to a change in the total protein content. Conclusion. A wide range of total protein concentrations may indicate a high adaptive potential of this organism. A decrease in the concentration of total protein may be due to the presence of a crustacean in unfavorable conditions and its defeat by a bacterial agent. It is noted that the change in protein concentration at different times of the year in a crustacean under artificial conditions cannot be interpreted as seasonal fluctuations.

An insect's living systems-circulation, respiration, and a branching nervous system-extend from the body into the wing. Wing hemolymph circulation is critical for hydrating tissues and supplying nutrients to living systems such as sensory organs across the wing. Despite the critical role of hemolymph circulation in maintaining healthy wing function, wings are often considered "lifeless" cuticle, and flows remain largely unquantified. High-speed fluorescent microscopy and particle tracking of hemolymph in the wings and body of the grasshopper Schistocerca americana revealed dynamic flow in every vein of the fore- and hindwings. The global system forms a circuit, but local flow behavior is complex, exhibiting three distinct types: pulsatile, aperiodic, and "leaky" flow. Thoracic wing hearts pull hemolymph from the wing at slower frequencies than the dorsal vessel; however, the velocity of returning hemolymph (in the hindwing) is faster than in that of the dorsal vessel. To characterize the wing's internal flow mechanics, we mapped dimensionless flow parameters across the wings, revealing viscous flow regimes. Wings sustain ecologically important insect behaviors such as pollination and migration. Analysis of the wing circulatory system provides a template for future studies investigating the critical hemodynamics necessary to sustaining wing health and insect flight.

The skin microbiome maintains healthy human skin, and disruption of the microbiome balance leads to inflammatory skin diseases such as folliculitis and atopic dermatitis. Staphylococcus aureus and Cutibacterium acnes are pathogenic bacteria that simultaneously inhabit the skin and cause inflammatory diseases of the skin through the activation of innate immune responses. Silkworms are useful invertebrate animal models for evaluating innate immune responses. In silkworms, phenoloxidase generates melanin as an indicator of innate immune activation upon the recognition of bacterial or fungal components. We hypothesized that S. aureus and C. acnes interact to increase the innate immunity-activating properties of S. aureus. In the present study, we showed that acidification is involved in the activation of silkworm hemolymph melanization by S. aureus. Autoclaved-killed S. aureus (S. aureus [AC]) alone does not greatly activate silkworm hemolymph melanization. On the other hand, applying S. aureus [AC] treated with C. acnes culture supernatant increased the silkworm hemolymph melanization. Adding C. acnes culture supernatant to the medium decreased the pH. S. aureus [AC] treated with propionic acid, acetic acid, or lactic acid induced higher silkworm hemolymph melanization activity than untreated S. aureus [AC]. S. aureus [AC] treated with hydrochloric acid also induced silkworm hemolymph melanization. The silkworm hemolymph melanization activity of S. aureus [AC] treated with hydrochloric acid was inhibited by protease treatment of S. aureus [AC]. These results suggest that acid treatment of S. aureus induces innate immune activation in silkworms and that S. aureus proteins are involved in the induction of innate immunity in silkworms. [ABSTRACT FROM AUTHOR]

This study aims to evaluate the effects of paprika extract on the survival rate, growth performance and stimulation of the innate immune system of Litopenaeus vannamei. In this experiment, 240 healthy shrimp (3.22 ± 0.12 g) were randomly divided into four groups. The shrimp were fed diets with different concentrations of paprika oil extracts (0%, 0.5%, 1% and 2%) for 8 weeks. The results showed that growth performance, urea, uric acid, creatinine, cholesterol levels, aspartate aminotransferase and alkaline phosphatase activities were not significantly affected by adding paprika extract to the shrimp diet (p > 0.05). Diets containing 1% and 0.5% paprika extract showed the highest levels of total protein and triglyceride, respectively (p < 0.05). There was a significant decrease in haemolymph glucose concentration in shrimp‐fed diets containing 1% and 2% paprika extract (p < 0.05). Moreover, a diet containing 0.5% paprika extract resulted in the highest levels of total heamocyte count, hyaline cells and large‐granular cells in shrimp (p < 0.05). Higher catalase and superoxide dismutase activities were also exhibited in the paprika groups (p < 0.05). Vibrio sp. bacteria were not significantly reduced by paprika extract in the intestines of L. vannamei (p > 0.05). A significant decrease in heterotrophic bacteria was observed with increasing extract concentrations (p < 0.05). The shrimp culture industry can utilize paprika extract as a cost‐effective, efficient and environmentally friendly immune stimulant at a concentration of 0.5%. [ABSTRACT FROM AUTHOR]

Due to some outstanding properties of dragonfly wings, such as a lightweight structure with good aerodynamic performance, they have attracted the attention of many researchers. The microscopic and macroscopic structures of dragonfly wings play important roles in their behaviors. One of these structures that are spread all over the wing is vein. In this study, as the first endeavour, the geometric characteristics of the vein are modeled precisely, and the natural frequencies of it are calculated. Next, the vibration of the vein with considering structural damping, stationary and moving hemolymph is investigated. In the cases where hemolymph is considered, the influences of fluid–structure interaction (FSI) of hemolymph on the vibrational behavior of wing vein are included. To show the accuracy of the used methodology, the present model is compared with the microscopic image of the vein. Besides, some comparisons with theoretical and experimental findings are also proposed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The invasion of bivalves by parasitic microalgae Coccomyxa is widespread and causes pathologies and dysfunctions of the organs, especially in the most valuable products: the mantle and the muscle. The pathogenesis of the disease remains completely unknown. In this study, based on a macroscopic examination of Modiolus kurilensis and microalgae count in each infected individual, four stages of disease development with characteristic pathognomonic symptoms were described. During the progression of the disease, the concentration of alkaline phosphatase, glucose, calcium, hemolytic and agglutinating activities, number of basophils, eosinophils, phagocytes, and cells with reactive oxygen species increased in the hemolymph, while number of agranulocytes, cells with lysosomes, dead hemocytes, total protein concentration, as well as the weight of mollusks decreased. In the nephridia and digestive gland, necrosis, invasion of Nematopsis sp., hemocyte infiltration, and fibrosis increased. The ratio of changed tubules and occurrence of granulocytomas increased in the digestive gland, while the base membrane, nephrocytes and concretions changed in the nephridia. This study helps establish the variability of these parameters under normal conditions and their alteration during the disease. Moreover, these findings can be used for veterinary monitoring of the state of bivalves in natural and aquaculture populations. [ABSTRACT FROM AUTHOR]

In insects vulnerable to dehydration, the mechanistic reaction of blood after wounding is rapid. It allows insects to minimize blood loss by sealing the wound and forming primary clots that provide scaffolding for the formation of new tissue. Using nano-rheological magnetic rotational spectroscopy with nickel nanorods and extensional rheology, we studied the properties of blood dripping from the wound of caterpillars of the Carolina sphinx moth (Manduca sexta) with a high concentration of blood cells. We discovered that wound sealing followed a two-step scenario. First, in a few seconds, the Newtonian lowviscosity blood turns into a non-Newtonian viscoelastic fluid that minimizes blood loss by retracting the dripping blood back into the wound. Next, blood cells aggregate, starting from the interfaces and propagating inward. We studied these processes using optical phase-contrast and polarized microscopy, X-ray imaging, and modeling. Comparative analyses of the cell-rich and cell-poor blood of different insects revealed common features of blood behavior. These discoveries can help design fast-working thickeners for vertebrate blood, including human blood. [ABSTRACT FROM AUTHOR]

Background: 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). Methodology/Principal findings: 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. Conclusions/Significance: 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. Author summary: Some factors can facilitate or prevent T. cruzi transmission, i.e. vector immunity. Our work has managed to detect a stronger immune response against T. cruzi II in R. prolixus saliva and haemolymph, compared to that of R. colombiensis. Proteins from both species' saliva and haemolymph were analysed for studying factors which might have been involved in such response; most proteins were detected in both species' haemolymph, thereby indicating common immune mechanisms. Three proteins having oxidative immune activity were only expressed in R. prolixus. Lipocalin diversity and abundance predominated in R. prolixus saliva; these proteins are involved in nitric oxide metabolism and their role in immunity could be key in host defence against T. cruzi. Recognising the components modulating parasite transmission in a vector helps in understanding how such factors act independently and how they would act synergistically against T. cruzi, thereby enabling us to establish tools regarding Chaga's disease epidemiology, aimed at predicting T. cruzi distribution and creating transmission control mechanisms. [ABSTRACT FROM AUTHOR]

The transmission of pathogens from reservoir to recipient host species, termed pathogen spillover, can profoundly impact plant, animal, and public health. However, why some pathogens lead to disease emergence in a novel species while others fail to establish or do not elicit disease is often poorly understood. There is strong evidence that deformed wing virus (DWV), an (+)ssRNA virus, spills over from its reservoir host, the honeybee Apis mellifera, into the bumblebee Bombus terrestris. However, the low impact of DWV on B. terrestris in laboratory experiments suggests host barriers to virus spread in this recipient host. To investigate potential host barriers, we followed the spread of DWV genotype B (DWV-B) through a host's body using RT-PCR after experimental transmission to bumblebees in comparison to honeybees. Inoculation was per os, mimicking food-borne transmission, or by injection into the bee's haemocoel, mimicking vector-based transmission. In honeybees, DWV-B was present in both honeybee faeces and haemolymph within 3 days of inoculation per os or by injection. In contrast, DWV-B was not detected in B. terrestris haemolymph after inoculation per os, suggesting a gut barrier that hinders DWV-B's spread through the body of a B. terrestris. DWV-B was, however, detected in B. terrestris faeces after injection and feeding, albeit at a lower abundance than that observed for A. mellifera, suggesting that B. terrestris sheds less DWV-B than A. mellifera in faeces when infected. Barriers to viral spread in B. terrestris following oral infection may limit DWV's impact on this spillover host and reduce its contribution to the community epidemiology of DWV. [ABSTRACT FROM AUTHOR]

Whiteleg shrimp (Litopenaeus vannamei) is a commercially important fishery commodity of high economic value. Intensive shrimp culture is done with high stocking density and large amount of feed requirement. Intensification can trigger stress, increase the possibility of physical contact among shrimps, and accelerate the spread of disease. The most common disease of shrimp is vibriosis that caused by Vibrio sp. However, shrimp has hemolymph, an innate immunity system that has an important role in body defense. This study was conducted from August to September 2023 using a purposive random sampling with 50 whiteleg shrimp samples. Assay of phagocytosis index (PI) that determines a cellular response of the shrimp body defense system was done at the Center for Brackish Water Aquaculture (BBPBAP) Jepara by applying a bacterial suspension of Staphylococcus sp. which has been attenuated, and bacterial staining was also carried out. Calculation of Total Vibrio Count (TVC) was done by using TCBS (Thiosulfate Citrate Bile Salt Sucrose Agar) agar media from Vibrio sp. bacteria cultured from the hepatopancreas of shrimp samples, and this activity was conducted at the Laboratory of Bacteriology and Mycology, Faculty of Veterinary Medicine, Universitas Airlangga. The results showed that whiteleg shrimp cultured in the intensive ponds in Situbondo was infected with Vibrio sp. with a TVC value of 3.3x105 cfu.ml-¹ and Phagocytosis index of 0.87. [ABSTRACT FROM AUTHOR]

Simple Summary: The silkworm is an economically important insect, and the popularity of artificial feed has promoted the development of the sericulture industry. However, the improvement of feed efficiency remains a challenge. This study used metabolomics-based methods to analyze the differences in silkworm cocoons and hemolymph between the mulberry-leaf group and the artificial-feed group. It was found that the concentration of amino acids was significantly higher in the mulberry-leaf group than in the artificial-diet group. In a comparison of the effects of different concentrations of valine on silkworms fed the artificial diet, it was found that the food intake of the 2% and 4% valine groups was significantly lower than that of the 0% valine group, but the cocoon-production efficiency was significantly improved. This study demonstrates that valine is an important amino acid that can regulate feed efficiency, providing a new direction for the improvement of artificial feed. Artificial silkworm diets significantly impact farm profitability. Sustainable cocoon production depends on the continuous improvement of feed efficiency to reduce costs and nutrient losses in the feed. This study used metabolomics to explore the differences in silkworm cocoons and hemolymph under two modes of rearing: an artificial diet and a mulberry-leaf diet. Nine metabolites of silkworm cocoons and hemolymph in the mulberry-leaf group were higher than those in the artificial-diet group. Enrichment analysis of the KEGG pathways for these metabolites revealed that they were mainly enriched in the valine, leucine, and isoleucine biosynthesis and degradation pathways. Hence, the artificial silkworm diet was supplemented various concentrations of valine were supplemented to with the aim of examining the impact of valine on their feeding and digestion of the artificial diet. The results indicated that valine addition had no significant effect on feed digestibility in the fifth-instar silkworm. Food intake in the 2% and 4% valine groups was significantly lower than that in the 0% valine group. However, the 2% and 4% valine groups showed significantly improved cocoon-production efficiency, at 11.3% and 25.1% higher, respectively. However, the cocoon-layer-production efficiencies of the 2% and 4% valine groups decreased by 7.7% and 13.9%, respectively. The research confirmed that valine is an effective substance for enhancing the feed efficiency of silkworms. [ABSTRACT FROM AUTHOR]

Simple Summary: The queen bee specializes in colony reproduction and undergoes behavioral and physical changes following egg-laying. Metabolomics is a high-throughput technique that can unveil the intricate connection between biological phenotypic states and an organism's small molecules. We are intrigued to determine the characteristic substances present in the hemolymph and ovary, as well as to understand how their rapid metabolism contributes to the process of egg-laying by queens. In this study, we reared Apis mellifera queens from three distinct time periods: newly emerged queen (NEQ), newly laying queen (NLQ), and old laying queen (OLQ). Using widely targeted metabolomics, we found that NLQ and OLQ exhibited significant metabolite alterations compared to NEQ, such as up-regulated expression of carnitines, fatty acids, and some antioxidants, and significantly down-regulated expression of amino acids, etc. The results of this study provide a novel perspective for analyzing the oviposition mechanism of queens. The queen bee is a central and pivotal figure within the colony, serving as the sole fertile female responsible for its reproduction. The queen possesses an open circulatory system, with her ovaries immersed in hemolymph. A continuous and intricate transportation and interchange of substances exist between the ovaries and hemolymph of queen bees. To determine the characteristic metabolites in the hemolymph and ovary, as well as understand how their rapid metabolism contributes to the process of egg-laying by queens, we reared Apis mellifera queens from three different age groups: newly emerged queen (NEQ), newly laying queen (NLQ), and old laying queen (OLQ). Using widely targeted metabolomics, our study revealed that the laying queen (NLQ and OLQ) exhibited faster fatty acid metabolism, up-regulated expression of antioxidants, and significant depletion of amino acids compared to the NEQ. This study revealed that the levels of carnitine and antioxidants (GSH, 2-O-α-D-glucopyranosyl-L-ascorbic acid, L-ascorbic acid 2-phosphate, etc.) in the NLQ and OLQ were significantly higher compared to NEQ. However, most of the differentially expressed amino acids, such as L-tryptophan, L-tyrosine, L-aspartic acid, etc., detected in NLQ and OLQ were down-regulated compared to the NEQ. Following egg-laying, pathways in the queens change significantly, e.g., Tryptophan metabolism, Tyrosine metabolism, cAMP signaling pathway, etc. Our results suggest that carnitine and antioxidants work together to maintain the redox balance of the queen. Additionally, various amino acids are responsible for maintaining the queen's egg production. [ABSTRACT FROM AUTHOR]

Simple Summary: Insect hemolymph transports, exchanges, and eliminates soluble compounds from the hemocoel. These processes play a critical role in many physiological processes, including development and immunity. Since matrix metalloproteases (MMPs) are the major circulating gelatinases in the blood of various species, including mammals, we used gel zymography to analyze gelatinase and caseinase activities in Drosophila larval hemolymph under normal and pathological conditions. Our investigations demonstrate that the major gelatinases and caseinases in Drosophila larval hemolymph are not MMPs but serine proteases (SPs). We identified more than 60 SPs in these proteolytic active bands. While a role in immunity has been suggested for some of these circulating SPs, the physiological functions of most of them remain to be elucidated. After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges. [ABSTRACT FROM AUTHOR]

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