Testing of arctic insect hemolymph as a secondary agent in applied cryopreservation.

BACKGROUND: Cold hardiness of insects from extremely cold regions is based on a principle of natural cryoprotection, which is associated with physiological mechanisms provided by cryoprotectants. OBJECTIVE: Since arctic cold-hardy insects are producers of highly effective cryoprotectants, in this study, the hemolymph of Aporia crataegi L. and Upis ceramboides L. from an extremely cold area (Yakutia) was tested as a secondary component of cryoprotective agents (CPA) for cryopreservation (-80 degree C) of human peripheral blood lymphocytes and skin fibroblasts. MATERIALS AND METHODS: Lymphocytes and skin fibroblasts were treated with various combinations of DMSO and hemolymph extract and step-wise cooled to -80 degree C. Post-cryopreservation cell viability was assessed by vital staining and morphological appearance. RESULTS: Viability was higher when cells were frozen with a mixture containing DMSO and Upis ceramboides hemolymph compared to the cells frozen in DMSO, while cells frozen with DMSO and Aporia crataegi hemolymph did not survive. The fact that hemolymph of not every cold-resistant insect can be used as a secondary agent along with DMSO indicates that only a unique combination of hemolymph components and its compatibility with cells might result in a positive effect. CONCLUSION: Although the use of insect hemolymph as a complementary agent in applied cryopreservation is a problem in terms of practical application, such studies could initiate new trends in the search for the most successful hemolymph-like cryoprotectant systems. https://doi.org/10.54680/fr24210110712.

Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

Feeding state-dependent modulation of feeding-related motor patterns.

Studies elucidating modulation of microcircuit activity in isolated nervous systems have revealed numerous insights regarding neural circuit flexibility, but this approach limits the link between experimental results and behavioral context. To bridge this gap, we studied feeding behavior-linked modulation of microcircuit activity in the isolated stomatogastric nervous system (STNS) of male Cancer borealis crabs. Specifically, we removed hemolymph from a crab that was unfed for ≥24 h ("unfed" hemolymph) or fed 15 min to 2 h before hemolymph removal ("fed" hemolymph). After feeding, the first significant foregut emptying occurred >1 h later and complete emptying required ≥6 h. We applied the unfed or fed hemolymph to the stomatogastric ganglion (STG) in an isolated STNS preparation from a separate, unfed crab to determine its influence on the VCN (ventral cardiac neuron)-triggered gastric mill (chewing) and pyloric (filtering of chewed food) rhythms. Unfed hemolymph had little influence on these rhythms, but fed hemolymph from each examined time-point (15 min, 1 h, or 2 h after feeding) slowed one or both rhythms without weakening circuit neuron activity. There were also distinct parameter changes associated with each time-point. One change unique to the 1-h time-point (i.e., reduced activity of one circuit neuron during the transition from the gastric mill retraction to protraction phase) suggested that the fed hemolymph also enhanced the influence of a projection neuron that innervates the STG from a ganglion isolated from the applied hemolymph. Hemolymph thus provides a feeding state-dependent modulation of the two feeding-related motor patterns in the C. borealis STG.NEW & NOTEWORTHY Little is known about behavior-linked modulation of microcircuit activity. We show that the VCN-triggered gastric mill (chewing) and pyloric (food filtering) rhythms in the isolated crab Cancer borealis stomatogastric nervous system were changed by applying hemolymph from recently fed but not unfed crabs. This included some distinct parameter changes during each examined post-fed hemolymph time-point. These results suggest the presence of feeding-related changes in circulating hormones that regulate consummatory microcircuit activity.

Real-time monitoring of biomechanical activity in aphids by laser speckle contrast imaging.

Studying in vivo feeding and other behaviors of small insects, such as aphids, is important for understanding their lifecycle and interaction with the environment. In this regard, the EPG (electrical penetration graph) technique is widely used to study the feeding activity in aphids. However, it is restricted to recording feeding of single insects and requires wiring insects to an electrode, impeding free movement. Hence, easy and straightforward collective observations, e.g. of groups of aphids on a plant, or probing other aphid activities in various body parts, is not possible. To circumvent these drawbacks, we developed a method based on an optical technique called laser speckle contrast imaging (LSCI). It has the potential for direct, non-invasive and contactless monitoring of a broad range of internal and external activities such as feeding, hemolymph cycling and muscle contractions in aphids or other insects. The method uses a camera and coherent light illumination of the sample. The camera records the laser speckle dynamics due to the scattering and interference of light caused by moving scatters in a probed region of the insect. Analyzing the speckle contrast allowed us to monitor and extract the activity information during aphid feeding on leaves or on artificial medium containing tracer particles. We present evidence that the observed speckle dynamics might be caused by muscle contractions, movement of hemocytes in the circulatory system or food flows in the stylets. This is the first time such a remote sensing method has been applied for optical mapping of the biomechanical activities in aphids.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication.

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host-pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host-pathogen transcriptomic landscape during systemic infection.

Effects of using the biofloc system and eyestalk ablation on reproductive performance and egg quality of Litopenaeus vannamei (Boone, 1931) (Decapoda: Dendrobranchiata: Penaeidae).

The aim of this study was to evaluate egg production and quality of females of the Pacific white shrimp, Litopenaeus vannamei, in which there was or was not unilateral eyestalk ablation after there was pre-maturation culturing in biofloc or clear-water systems. Acylglycerides, cholesterol, glucose and total soluble protein were determined for the hepatopancreas, ovaries, hemolymph and eggs. Females cultured using the biofloc system had a larger number of eggs released per spawning and per gram of spawning specimen body weight. The number of total spawning's per week was comparable among treatments. Females cultured in the biofloc system in which there was no eyestalk ablation had that greatest concentrations of nutrient reserves in the hepatopancreas (P < 0.05) with the females cultured in the biofloc and clear-water system that had eye stalk ablation having the next most abundant nutrient reserves. There were the least concentrations of nutrient reserves in females with eyestalk ablation cultured in the clear-water system (P < 0.05). There, however, were no difference in nutrient reserve concentrations in the hemolymph and ovaries. In the eggs, there was the same trend among treatments as the hepatopancreas nutrient reserves, indicating that both eyestalk ablation and pre-maturation culture conditions (i.e., either biofloc or clear-water) affected the quality of eggs in L. vannamei.

Exploring the versatility of the perfused crustacean gill as a model for transbranchial transport processes.

The study of transbranchial ion and gas transport of water-breathing animals has long been a useful means of modeling transport processes of higher vertebrate organs through comparative physiology. The molecular era of biological research has brought forward valuable information detailing shifts in gene expression related to environmental stress and the sub-cellular localization of transporters; however, purely molecular studies can cause hypothetical transport mechanisms and hypotheses to be accepted without any direct physiological proof. Isolated perfused gill experiments are useful for testing most of these hypotheses and can sometimes be used outright to develop a well-supported working model for transport processes relating to an animal's osmoregulation, acid-base balance, nitrogen excretion, and respiratory gas exchange as well as their sensitivity to pollutants and environmental stress. The technique allows full control of internal hemolymph-like saline as well as the ambient environmental fluid compositions and can measure the electrophysiological properties of the gill as well as the transport rates of ions and gases as they traverse the gill epithelium. Additives such as pharmaceuticals or peptides as well as the exclusion of ions from the media are commonly used to identify the importance of specific transporters to transport mechanisms. The technique can also be used to identify the penetrance, retention, and localization of pollutants within the gill epithelium or to explore the uptake and metabolism of nutrients directly from the ambient environment. While this technique can be applied to virtually any isolatable organ, the anatomy and rigidity of the decapod crustacean gill make it an ideal candidate for most experimental designs.

Mating experience modifies locomotor performance and promotes episodic motor activity in Drosophila melanogaster.

Sexual behavior is a routine among animal species. Sexual experience has several behavioral consequences in insects, but its physiological basis is less well-understood. The episodic motor activity with a periodicity around 19 s was unintentionally observed in the wildtype Canton-S flies and was greatly reduced in the white-eyed mutant w1118 flies. Episodic motor activity co-exists with several consistent locomotor performances in Canton-S flies whereas reduced episodic motor activity is accompanied by neural or behavioral abnormalities in w1118 flies. The improvements of both episodic motor activity and locomotor performance are co-inducible by a pulsed light illumination in w1118. Here we show that mating experience of w1118 males promoted fast and consistent locomotor activities and increased the power of episodic motor activities. Compared with virgin males, mated ones showed significant increases of boundary preference, travel distance over 60 s, and increased path increments per 0.2 s. In contrast, mated males of Canton-S showed decreased boundary preference, increased travel distance over 60 s, and increased path increments per 0.2 s. Additionally, mated males of w1118 displayed increased power amplitude of periodic motor activities at 0.03-0.1 Hz. These data indicated that mating experience promoted fast and consistent locomotion and improved episodic motor activities in w1118 male flies.

The circulatory system of Penaeus vannamei Boone, 1931-Lacunar function and a reconsideration of the "open vs. closed system" debate.

The morphology of hemolymph circulatory systems has been studied in many arthropod groups over the past decades. In most cases, however, the focus of these studies has been the vascular system, while its counterpart, the lacunar system, has often been neglected. To further understanding of the interrelationships between these two complementary subsystems, we investigated both, the hemolymph vascular system and the hemolymph lacunar system, of the decapod Penaeus vannamei using 3D-imaging techniques (micro-computed tomography and confocal laser scanning microscopy) in combination with 3D reconstruction. Major parts of the vascular and lacunar system are described. Our insights into their morphology are used to derive functional conclusions for a model illustrating the interrelationships between the two subsystems. The morphology of and the functional interaction between the vascular and lacunar systems are discussed in the context of the debate on "open vs. closed circulatory systems."

Physical and behavioral adaptations to prevent overheating of the living wings of butterflies.

The wings of Lepidoptera contain a matrix of living cells whose function requires appropriate temperatures. However, given their small thermal capacity, wings can overheat rapidly in the sun. Here we analyze butterfly wings across a wide range of simulated environmental conditions, and find that regions containing living cells are maintained at cooler temperatures. Diverse scale nanostructures and non-uniform cuticle thicknesses create a heterogeneous distribution of radiative cooling that selectively reduces the temperature of structures such as wing veins and androconial organs. These tissues are supplied by circulatory, neural and tracheal systems throughout the adult lifetime, indicating that the insect wing is a dynamic, living structure. Behavioral assays show that butterflies use wings to sense visible and infrared radiation, responding with specialized behaviors to prevent overheating of their wings. Our work highlights the physiological importance of wing temperature and how it is exquisitely regulated by structural and behavioral adaptations.

A comparison of low temperature biology of Pieris rapae from Ontario, Canada, and Yakutia, Far Eastern Russia.

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. -9.3 °C and were freeze-tolerant in 2002-2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< -20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to -15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

Physiological impacts of time in holding ponds, biomedical bleeding, and recovery on the Atlantic horseshoe crab, Limulus polyphemus.

Atlantic horseshoe crabs, Limulus polyphemus (HSC), are commercially harvested along the eastern U.S. coast and bled for hemolymph used in pharmaceutical safety testing. In South Carolina, some HSCs are held in outdoor ponds before transport to facilities where they are bled and then released to the wild. This study examined whether the time HSCs are held before bleeding, bleeding itself, or the duration of the recovery after bleeding affects HSC mortality and physiological condition. Female HSCs were collected from Coffin Point Beach, South Carolina (April 22-24, 2016), held in ponds for 2, 4, 6, or 8 weeks, then bled or held as controls. Body weights, hemocyanin concentrations, and hemocyte densities were measured prior to treatment (bled/control) and at 2, 6, and 12 days of recovery. Hemocyanin concentrations declined significantly in HSCs held in ponds for 8 weeks prior to bleeding and were excluded from further analyses. Compared to some studies, HSC mortalities were low (11%). Impacts of time in holding ponds, bleeding, and recovery from bleeding on physiological measures were assessed using 3-way fixed-effects ANOVA. While duration of recovery had main effects on physiological measures, significant interactions were also present. There was an interaction of treatment and recovery duration, with control crabs having higher hemocyte densities than bled animals at days 2 and 6 of recovery. There were two significant two-way interactions influencing hemocyanin concentration: pond time and recovery, and treatment and recovery. Our study suggests both main and synergistic effects are important when assessing the physiology and mortality of HSCs harvested for biomedical purposes.

The Insect Circulatory System: Structure, Function, and Evolution.

Although the insect circulatory system is involved in a multitude of vital physiological processes, it has gone grossly understudied. This review highlights this critical physiological system by detailing the structure and function of the circulatory organs, including the dorsal heart and the accessory pulsatile organs that supply hemolymph to the appendages. It also emphasizes how the circulatory system develops and ages and how, by means of reflex bleeding and functional integration with the immune system, it supports mechanisms for defense against predators and microbial invaders, respectively. Beyond that, this review details evolutionary trends and novelties associated with this system, as well as the ways in which this system also plays critical roles in thermoregulation and tracheal ventilation in high-performance fliers. Finally, this review highlights how novel discoveries could be harnessed for the control of vector-borne diseases and for translational medicine, and it details principal knowledge gaps that necessitate further investigation.

Clathrin-dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori.

During insect larval-pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph-decreased protein bands (55-100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin-dependent endocytosis predominantly blocked the transportation of 55-100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ∼90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph-specific proteins were mainly involved in material transportation, while fat body-specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin-dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.

Osmoionic homeostasis in bivalve mollusks from different osmotic niches: Physiological patterns and evolutionary perspectives.

Physiological knowledge gained from questions focused on the challenges faced and strategies recruited by organisms in their habitats assumes fundamental importance about understanding the ability to survive when subjected to unfavorable situations. In the aquatic environment, salinity is particularly recognized as one of the main abiotic factors that affects the physiology of organisms. Although the physiological patterns and challenges imposed by each occupied environment are distinct, they tend to converge to osmotic oscillations. From a comparative perspective, we aimed to characterize the osmoregulatory patterns of the bivalve mollusks Corbicula largillierti (purple Asian cockle), Erodona mactroides (lagoon cockle), and Amarilladesma mactroides (white clam) - inhabitants of different osmotic niches - when submitted to hypo- and/or hyperosmotic salinity variations. We determined the hemolymph osmotic and ionic concentrations, tissue hydration, and the intracellular isosmotic regulation (IIR) from the use of osmolytes (organic and inorganic) after exposure to species-specific salinity intervals. Additionally, we incorporated phylogenetic perspectives to infer and even broaden the understanding about the patterns that comprise the osmoionic physiology of Bivalvia representatives. According to the variables analyzed in the hemolymph, the three species presented a pattern of osmoconformation. Furthermore, both ionic regulation and conformation patterns were observed in freshwater, estuarine, and marine species. The patterns verified experimentally show greater use of inorganic osmolytes compared to the participation of organic molecules, which varied according to the osmotic niche occupied in the IIR for the mantle, adductor muscle, and gills. This finding widens the classic vision about the preferential use of certain osmolytes by animals from distinct niches. Our phylogenetic perspective also indicates that environmental salinity drives physiological trait variations, including hemolymph osmolality and the ion composition of the extracellular fluid (sodium, chloride, magnesium, and calcium). We also highlight the important role played by the shared ancestry, which influences the interspecific variability of the hemolymph K+ in selected representatives of Bivalvia.

Effects of the Biomedical Bleeding Process on the Behavior of the American Horseshoe Crab, Limulus polyphemus, in Its Natural Habitat.

Horseshoe crabs are harvested by the biomedical industry in order to create Limulus amebocyte lysate to test medical devices and pharmaceutical drugs for endotoxins. Most previous studies on the impacts of the biomedical bleeding process on horseshoe crabs have focused on mortality rates and sublethal impacts in the laboratory. In this study, we investigated the effects of the bleeding process on the behavior of horseshoe crabs after they had been released back into their natural environment. A total of 28 horseshoe crabs (14 control and 14 bled) were fitted with acoustic transmitters and released into the Great Bay Estuary, New Hampshire, during the spring of 2016. The acoustic tags transmitted information about the activity and depth of each animal, and these data were logged by an array of passive acoustic receivers. These data were collected from May to December 2016 and from March to October 2017. Bled animals approached mating beaches less than control animals during the first week after release, with the greatest differences between bled and control females. Bled animals also remained significantly deeper during the spawning season than control animals. However, overall, bled and control animals expressed similar biological rhythms and seasonal migrations. Thus, it appears as if the most obvious impacts of the bleeding process take place during the first one to two weeks after crabs are bled.

Functional compartmentalization in the hemocoel of insects.

The insect circulatory system contains an open hemocoel, in which the mechanism of hemolymph flow control is ambiguous. As a continuous fluidic structure, this cavity should exhibit pressure changes that propagate quickly. Narrow-waisted insects create sustained pressure differences across segments, but their constricted waist provides an evident mechanism for compartmentalization. Insects with no obvious constrictions between segments may be capable of functionally compartmentalizing the body, which could explain complex hemolymph flows. Here, we test the hypothesis of functional compartmentalization by measuring pressures in a beetle and recording abdominal movements. We found that the pressure is indeed uniform within the abdomen and thorax, congruent with the predicted behavior of an open system. However, during some abdominal movements, pressures were on average 62% higher in the abdomen than in the thorax, suggesting that functional compartmentalization creates a gradient within the hemocoel. Synchrotron tomography and dissection show that the arthrodial membrane and thoracic muscles may contribute to this dynamic pressurization. Analysis of volume change suggests that the gut may play an important role in regulating pressure by translating between body segments. Overall, this study suggests that functional compartmentalization may provide an explanation for how fluid flows are managed in an open circulatory system.

Insect hemolymph coagulation: Kinetics of classically and non-classically secreted clotting factors.

In most insects, hemolymph coagulation, which is analogous to mammalian blood clotting, involves close collaboration between humoral and cellular components. To gain insights into the secretion of cellular clotting factors, we created tagged versions of three different clotting factors. Our focus was on factors which are released in a non-classical manner and to characterize them in comparison to a protein that is classically released, namely Glutactin (Glt). Transglutaminase-A (Tg) and Prophenoloxidase 2 (PPO2), both of which lack signal peptide sequences, have been previously demonstrated to be released from plasmatocytes and crystal cells (CCs) respectively, the two hemocyte classes in naïve larvae. We found that at the molecular level, Tg secretion resembles the release of tissue transglutaminase in mammals. Specifically, Drosophila Tg is associated with vesicular membranes and remains membrane-bound after release, in contrast to Glt, which we found localizes to a different class of vesicles and is integrated into clot fibers. PPO2 on the other hand, is set free from CCs through cytolysis. We confirm that PPO2 is a central component of the cytosolic crystals and find that the distribution of PPO2 appears to vary across crystals and cells. We propose a tentative scheme for the secretory events during early and late hemolymph coagulation.

The art of a hydraulic joint in a spider's leg: modelling, computational fluid dynamics (CFD) simulation, and bio-inspired design.

Important aspects of spider locomotion rely on a hydraulic mechanism. So far, this has not been theoretically analysed. In this work, the flow mechanism of a main hydraulic joint in a spider leg was studied. The purpose is to gain insight into a biohydraulic mechanism using an engineering approach to improve our understanding of the hemolymph flow path in the spider's legs and to contribute to the theoretical analysis of the spider's hydraulic transmission mechanism, thereby providing an inspiration for advanced biomimetic hydraulic systems. During the study, Micro-CT results were used to reconstruct the detailed flow channel. The high-pressure areas (inlet, joint, and closed leg end) and low pressures in between are also identified. Then, the internal flow field was investigated using computational fluid dynamics. At the same time, the method of dynamic mesh regeneration, elastic smoothing, is used to simulate muscle contraction and joint extension. The different functions of the channels are substantiated by the velocity profiles. Finally, a bionic hydraulic system was designed according to the trajectory of haemolymph in the flow channel.

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model.

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses.