Viscosity, which impacts the rate of haemolymph circulation and heat transfer, is one of the transport properties that affects the performance of an insect. Measuring the viscosity of insect fluids is challenging because of the small amount available per specimen. Using particle tracking microrheology, which is well suited to characterise the rheology of the fluid part of the haemolymph, we studied the plasma viscosity in the bumblebee Bombus terrestris. In a sealed geometry, the viscosity exhibits an Arrhenius dependence with temperature, with an activation energy comparable to that previously estimated in hornworm larvae. In an open to air geometry, it increases by 4–5 orders of magnitude during evaporation. Evaporation times are temperature dependent and longer than typical insect haemolymph coagulation times. Unlike standard bulk rheology, microrheology can be applied to even smaller insects, paving the way to characterise biological fluids such as pheromones, pad secretions or cuticular layers.

Haemolymph, the counterpart of blood in invertebrates, circulates freely throughout the insect body, bathing all internal tissues. It is composed of a fluid plasma in which are suspended haemocytes, cellular defence units partly responsible for the immune system of insects (Siddiqui and Khalifa, 2014; Kanost, 2009). Plasma also contains many compounds such as free amino acids or proteins which differ from one insect to another, and potentially between females and males during development. Sugars, such as trehalose, whose function is to provide energy to tissues, are also present (Wyatt, 1961; Chapman et al., 2013; Hillyer and Pass, 2020). Haemolymph plays a major role in the survival and physiology of insects. This circulating fluid transports nutrients, hormones and waste products through the body, maintaining correct humidity, optimal body temperature and body shape (Chapman et al., 2013; Nation, 2016). In addition, when the cuticle is damaged, haemolymph coagulates, preventing pathogens from entering the insect body (Bidla et al., 2005; Theopold et al., 2004).

Among the physical properties that govern the flow of a fluid, viscosity is a key parameter, and it depends on temperature. Unlike mammals, the body temperature of insects varies according to activity and the temperature of the external environment by thermoregulation (Heinrich, 1995). These temperature variations directly affect the viscosity of plasma and subsequently haemolymph, and therefore their circulation. Such changes in viscosity have important consequences for insect physiology. Several studies show that, among living beings, insects suffer particularly from the increase in temperature and temperature extremes induced by global warming (Heinrich, 1995; Halsch et al., 2021; Liebhold and Bentz, 2011; Robinet and Roques, 2010). In addition, studies show that heat waves could have an impact on the body water content of insects such as bumblebees (Burdine and McCluney, 2019; Chown et al., 2011). They would therefore have a direct effect on the viscosity of the plasma of the insect. In this context, probing the influence of temperature on the rheological properties of haemolymph and plasma appears to be crucial.

Because of their small size, insects contain small volumes of fluid. This makes it difficult to collect the fluid and analyse it (Tabunoki et al., 2019). Recently, the rheology of plasma and haemolymph has been studied in lepidoptera larvae, but not in adult insects (Kenny et al., 2018; Aprelev et al., 2019). Kenny et al. (2018) measured the viscosity in hornworm larvae with a cone and plate viscometer using a volume of 0.7 ml per sample per specimen. These large quantities of fluid can only be collected from large insects, which represent a small fraction of insect species. In general, once the liquid extraction procedure has been implemented, the volume obtained for most insects is too small to perform viscosity measurements with a standard rheometer.

In this context, particle tracking microrheology appears to be a relevant alternative technique. It typically requires 1 µl of fluid and consists of determining the fluid rheological properties from the Brownian motion of micrometric tracer beads immersed in it. At the micrometre scale, the beads probe the relationship between stress and deformation in their local environment. The technique is well suited to the study of small insects that secrete very small amounts of fluid, provided that it can be extracted, but also to the study of biological entities, such as living cells (Waigh, 2005, 2016). Recent studies on insect footprints or on the cuticular hydrocarbon layer of ants have shown that it is even possible to carry out viscosity measurements with sample volumes of the order of 10 pl (Abou et al., 2010; Sprenger et al., 2018; Menzel et al., 2019).

In this paper, we present measurements of the plasma viscosity of the common buff-tailed bumblebee Bombus terrestris with temperature, using particle tracking microrheology. This technique is particularly useful for the study of the rheological properties of the plasma. In the haemolymph, however, the tracer beads would only probe the suspension medium of the blood cells, the plasma, and not the haemolymph, because the distance between the haemocytes is greater than the size of the tracer beads (1 µm in diameter). A different technique is therefore needed to study the rheology of the whole fluid, the plasma and the blood cells, such as the one used to study hornworm haemolymph (Kenny et al., 2018), which requires larger sample volumes. However, an interesting aspect of the microrheology technique used here for haemolymph is that it can detect spatial heterogeneities in the rheology of the material by analysis of different tracers localised in the sample. It could therefore be used to detect haemolymph coagulation by observing beads immersed in different areas of the sample, whether liquid or gel. This technique could also complement recent measurements using nanorheological magnetic rotational spectroscopy with nanorods, which has allowed the quantitative study of nucleation of cell aggregates during coagulation (Aprelev et al., 2019). In this active microrheology technique, a magnetic torque is applied to 10 µm long nanorods. The response of the material, probed at the nanorod scale, can be probed in the non-linear large deformation regime, and may depend on the amplitude of the magnetic torque. In the clot, for example, the nanorod may destroy the structure under a significant torque and the analysis of the rheological properties may not be straightforward. With our particle tracking technique, in contrast, only the linear regime of small deformation is probed, by thermal agitation of the tracer beads. As they provide information in different deformation regimes and at different length scales, the two techniques are complementary.

Our efforts focused on bumblebees because, among the declining insects, they play a crucial role in the pollination of crops and wild plants in temperate regions and are particularly sensitive to temperature increases (Heinrich, 2004; Soroye et al., 2020; Martinet et al., 2021). The rheology of the bumblebee plasma was studied in two geometries, one open to air and the other sealed. In the sealed geometry, which prevents contamination and evaporation, we compared the plasma viscosity of bumblebees B. terrestris with that of hornworm larvae measured by Kenny et al. (2018). In open geometry, we measured the evaporation dynamics of the plasma drop as a function of temperature, as well as the associated characteristic evaporation times.

Bumblebees

Bumblebees, Bombus terrestris (Linnaeus 1758), were obtained from the standard product Masculino system (Bombus terrestris) from Biobest Group NV (Westerlo, Belgium). Only male specimens were selected because their plasma composition is less variable over time than that of females, whose hormonal state varies (Heinrich, 2004). Specimens were grouped by 50 in a cardboard box, were all the same age (3–4 days) and were fed with liquid sugar supplied by Biobest (Biosweet). Plasma was collected within a week of receipt.

Extraction of haemolymph and preparation of plasma samples

A protocol adapted from Arafah et al. (2019) was used to extract haemolymph from bumblebees. It was collected using a stretched glass capillary with a large tip (external diameter ∼50–200 µm), inserted a few millimetres into the insect body to avoid contamination. The fluid was drawn up by capillary effect by placing the glass capillary dorsally under the second tergite of the abdomen (Fig. 1A). Puncturing the dorsal region allows a sufficient quantity of fluid to be collected without injuring the bumblebees. It also avoids perforating the digestive tract, which would be a source of contamination for the sample. Volumes of 20 µl per individual were collected. They were visually clear, providing further confirmation of the absence of contamination from fatty substances, which would have caused a slight haze to form. Batches of fluid from five bumblebees were prepared, dispensed into Eppendorf tubes and sealed under argon gas. In practice, the fluid obtained using this protocol is mainly plasma, with only a few cells suspended. The samples were then left to sediment in the sealed Eppendorf tubes for a few tens of minutes (∼30–100 min) in order to separate the plasma from the few cells present. The supernatant – corresponding to the plasma – was then collected and distributed in 5 µl Eppendorf tubes sealed under argon gas. A small volume of a concentrated aqueous suspension of melamine beads (bead volume fraction 10%, bead diameter 0.88 µm; Granuloshop, Chatou, France) was added to the different plasma batches. The ratio of bead suspension volume to plasma was calculated to give a volume fraction of beads of less than 1% in each batch. The different batches were stored at 20°C for up to 2 months (Fig. 1A). Mixing five bumblebee samples allowed us to test the suitability of the technique independently of biological variability, while measuring the general behaviour of bumblebee plasma and limiting the bias associated with variations in composition between individuals. However, the same approach could have been used to measure each sample from the same individual independently.

Fig. 1.

Particle tracking microrheology technique. (A) Principle of plasma extraction from bumblebees and microrheology measurements. The plasma is collected through a stretched capillary tube inserted into the insect body. Plasma samples from 5 specimens are mixed with a small volume of a concentrated aqueous suspension of melamine beads. This mixture is then distributed into 5 µl Eppendorf tubes. (B). Sketch of the sealed geometry (left) and the open geometry (right) configurations. A drop of plasma/beads is placed on the microscope slide (1); in the sealed configuration, this is then covered by a coverslip (2). (C) Calibration curves of the sample temperature, ΔTdrop (difference between the set temperature and the sample temperature), and the objective temperature, ΔTobj (difference between the set temperature and the objective temperature), as a function of the room temperature ΔTroom (difference between the set temperature and the room temperature). The shaded areas highlight the temperature ranges measured with a drop of water as a sample. (D) Tracking of beads over 5 s (sealed geometry at 30°C, η=1.7 mPa s). The colour associated with the position of the bead varies over time (from yellow to blue). Scale bar: 10 µm. Inset: magnification of the displacement of a bead (scale bar: 1 µm).

Fig. 1.

Particle tracking microrheology technique. (A) Principle of plasma extraction from bumblebees and microrheology measurements. The plasma is collected through a stretched capillary tube inserted into the insect body. Plasma samples from 5 specimens are mixed with a small volume of a concentrated aqueous suspension of melamine beads. This mixture is then distributed into 5 µl Eppendorf tubes. (B). Sketch of the sealed geometry (left) and the open geometry (right) configurations. A drop of plasma/beads is placed on the microscope slide (1); in the sealed configuration, this is then covered by a coverslip (2). (C) Calibration curves of the sample temperature, ΔTdrop (difference between the set temperature and the sample temperature), and the objective temperature, ΔTobj (difference between the set temperature and the objective temperature), as a function of the room temperature ΔTroom (difference between the set temperature and the room temperature). The shaded areas highlight the temperature ranges measured with a drop of water as a sample. (D) Tracking of beads over 5 s (sealed geometry at 30°C, η=1.7 mPa s). The colour associated with the position of the bead varies over time (from yellow to blue). Scale bar: 10 µm. Inset: magnification of the displacement of a bead (scale bar: 1 µm).

Microrheology

Particle tracking microrheology is a technique that consists of determining the rheological properties of a material from the study of the Brownian motion of micrometric beads embedded in the material (for reviews, see Waigh, 2005, 2016). In our setup, the sample was placed under an inverted microscope (Leica DM IRB, Germany) at ×100 magnification (oil immersion, Numerical Aperture 1.3), and the Brownian motion of the micrometric beads was measured with a high-speed sCMOS camera (EoSens Mikrotron, Photon Lines, Pacé, France). Below, we describe the two working geometries in which the samples were studied, the temperature control system of the sample, and finally the analysis of the Brownian motion, which enables the determination of the rheological properties of the material.

Working geometry and temperature control

Two working geometries, referred to as sealed and open, were used to study the rheological properties of the plasma. In the sealed geometry, which prevents contamination or evaporation, a volume of 2.5 μl of plasma was deposited on a microscope slide and a microscope coverslip was placed over the top, separated by a thin adhesive spacer of dimension 1 cm×1 cm×250 μm (Gene frame®, VWR International) (Fig. 1B, left). In the open geometry, a 3 μl drop of plasma was deposited on a microscope slide and left in the open air (Fig. 1B, right). Before depositing the plasma drop in the working geometry, the Eppendorf tubes were thawed at room temperature and stirred for a few seconds to disperse the beads evenly throughout the sample.

The microscope objective was equipped with a laboratory-designed heating ring which includes a water circulation controlled by a thermostat to within ±0.3°C. The temperature of the plasma drop could thus be adjusted through the objective heating ring via the immersion oil in contact with the sample, as shown in Fig. 1B. A peristaltic micro-pump ensured a continuous exchange of water between the thermostat and the heating ring.

To predict the temperature of our plasma droplet samples with our device, calibration curves were established with water droplets whose temperature was directly measured with an internally positioned thermocouple probe. Fig. 1C displays the evolution of ΔTobj (=TsetTobj) and ΔTdrop (=TsetTdrop) with ΔTroom (=TsetTroom), where Tset is the temperature of the thermostatic bath, Tobj is the temperature of the objective measured just below the sample and Tdrop is the temperature of a water droplet (Fig. 1B). These calibration experiments allowed us to establish the following calibration laws to be used with the plasma droplets in the microrheology experiments: ΔTdrop=0.65ΔTroom±1.21°C and ΔTobj=0.51ΔTroom±0.52°C.

Brownian motion analysis

Once the plasma droplet was deposited in the working geometry and thermalisation was effective, the Brownian motion of the tracer beads in the droplet was recorded for 20 s at 100 frames s−1 with the high-speed camera under the inverted microscope. The positions xi(t) and yi(t) of each bead near the focal plane (typically between 15 and 30 beads depending on the realisation) were tracked with image analysis software developed locally under ImageJ (https://imagej.nih.gov/ij). The beads close to the surfaces – the microscope slide and the free surface of the drop – were not selected in order to avoid measurement bias due to the friction of the probe near a surface. For each tracer bead i, the time-averaged mean-squared displacement was calculated from the successive positions xi and yi between time t and t′, according to . The ensemble-averaged mean-squared displacement (MSD), ⟨Δr2(t)⟩, was calculated as the average of the time-averaged MSDs over all beads i in the sample. The ensemble-averaged MSD is the relevant quantity that allows us to extract the rheological properties of the material from the Brownian motion of the beads (Waigh, 2005, 2016; Einstein, 1905).

In a purely viscous fluid of viscosity η, such as water or glycerol, the Brownian motion of the probes is diffusive. This means that the ensemble-averaged MSD linearly increases with time, according to (in two dimensions), where D is the diffusion coefficient. In this simple case, the viscosity η can be estimated using the Stokes–Einstein relation, η=kT/6πRD, where R is the bead radius and kT is the thermal energy (Einstein, 1905). A number of soft materials (foams, colloids, gels), however, display more complex rheological properties than simple fluids. This is the case for viscoelastic fluids, which are both viscous and elastic on the time scale at which they are probed. In that case, the ensemble-averaged MSD adopts a dependence that is no longer linear with time, which is a signature of the fact that the probed material is viscoelastic. In such cases, a generalized Stokes–Einstein equation will be used to determine the elastic and viscous moduli of the probed material (Waigh, 2005, 2016; Mason, 2000).

Plasma viscosity in sealed and open geometry

Plasma droplet viscosity was measured in open and sealed geometry as a function of waiting time tw – the time since the droplet was deposited on the substrate. Fig. 2A–E shows the MSD for each bead in the sample (grey symbols), as well as the ensemble-averaged MSD (average over all beads in the sample, coloured symbols), as a function of waiting time tw, at a temperature 15°C. In both geometries and at any tw, the ensemble-averaged MSD was linear with time. This indicates that the plasma is a purely viscous fluid, and can be described by a single parameter, the viscosity η. From the ensemble-averaged MSDs in Figs 2A–E, we extracted a diffusion coefficient D, according to , and then a viscosity η inversely proportional to D, according to the Stokes–Einstein relation (Einstein, 1905). Fig. 2F shows the viscosity in the two geometries as a function of tw. While remaining constant in the sealed geometry, the viscosity increased by 4–5 orders of magnitude over 100 min in the open geometry, where water evaporation takes place. A close examination of our MSD data shows that the plasma remains a spatially homogeneous fluid at the bead scale – with or without evaporation – as demonstrated by the fact that the different beads in the sample (grey symbols) lead to a similar time dependence of the MSD for each tw and geometry.

Fig. 2.

Plasma viscosity in the sealed and open geometry. (A–E) Mean-squared displacement (MSD) of micrometric melamine beads (0.88 µm in diameter) in a plasma drop, in open (circles) and sealed (triangles) geometry, at waiting times tw=5 min, tw=15 min, tw=25 min, tw=35 min and tw=45 min. The temperature was set at 15°C. The grey symbols correspond to individual beads, while the coloured symbols correspond to the average of all beads in the sample (17–33 beads depending on the realisation). In both geometries and at any tw, the ensemble-averaged MSD is linear with time, indicating that the plasma is a purely viscous liquid. (F) Viscosity versus waiting time in both geometries, at 15°C. In the open geometry, the viscosity increases by 4–5 orders of magnitude over 100 min, while it remains constant in the sealed geometry. The coloured vertical lines correspond to the different tw in graphs A–E.

Fig. 2.

Plasma viscosity in the sealed and open geometry. (A–E) Mean-squared displacement (MSD) of micrometric melamine beads (0.88 µm in diameter) in a plasma drop, in open (circles) and sealed (triangles) geometry, at waiting times tw=5 min, tw=15 min, tw=25 min, tw=35 min and tw=45 min. The temperature was set at 15°C. The grey symbols correspond to individual beads, while the coloured symbols correspond to the average of all beads in the sample (17–33 beads depending on the realisation). In both geometries and at any tw, the ensemble-averaged MSD is linear with time, indicating that the plasma is a purely viscous liquid. (F) Viscosity versus waiting time in both geometries, at 15°C. In the open geometry, the viscosity increases by 4–5 orders of magnitude over 100 min, while it remains constant in the sealed geometry. The coloured vertical lines correspond to the different tw in graphs A–E.

Temperature dependence of viscosity

Plasma viscosity was first measured in the sealed geometry between 12.5 and 32.5°C. The temperature dependence is shown in Fig. 3A where the viscosity is plotted as a function of the inverse temperature 1/T. For comparison, viscosity data in hornworm plasma obtained with standard rheology (Kenny et al., 2018), as well as tabulated viscosity–temperature data in water (Rumble, 2021), are reported. The plasma viscosity of the bumblebee was higher than that of the hornworm and that of water. It decreased by a factor of 2 per temperature increase between 12.5 and 32.5°C. The temperature dependence of viscosity could be described using a classical Arrhenius model for viscous fluids, η(T)=ηexp(Ea/RT), where η is a reference viscosity, R is the gas constant per mole (8.34 J mol1 K1) and Ea is the Arrhenius activation energy (De Guzman, 1913; Eyring, 1935, 1936). For the bumblebee plasma, Ea was estimated to be 22.1 kJ mol1, comparable to, although larger than, that estimated in the hornworm larvae plasma, Ea=18.2±2.2 kJ mol1, from the data of Kenny et al. (2018). The Ea of water was estimated to be 18.0±1.1 kJ mol1, lower than that obtained for insects. This is consistent with the fact that insect plasma is composed of 90% water, but also other components such as highly soluble organic and inorganic constituents. It is interesting here to consider that the difference between the activation energies of the insect plasmas is related to their difference in composition, and is a signature of the species considered.

Fig. 3.

Temperature dependence of viscosity. (A) Viscosity of bumblebee plasma in sealed geometry as a function of inverse temperature. For comparison, viscosity is also shown for water (Rumble, 2021) and hornworm plasma (Kenny et al., 2018). Data are means±s.d. The dashed lines represent the respective fit according to Arrhenius law. (B) Viscosity of bumblebee plasma in open geometry in the range 15–30°C as a function of waiting time (the inset shows a log-linear plot of the data). The constant viscosity values obtained in the sealed geometry for the same temperatures have been added (horizontal dashed coloured lines). Five experiments were performed per temperature, except at 15°C, where three experiments were performed. (C) Characteristic evaporation time te, defined for a viscosity of 1 Pa s (horizontal dashed black line in B), as a function of temperature. Evaporation time decreases with temperature, according to te=t0exp(−T/T1), where t0=144.9 min and T1=14.1°C (solid line).

Fig. 3.

Temperature dependence of viscosity. (A) Viscosity of bumblebee plasma in sealed geometry as a function of inverse temperature. For comparison, viscosity is also shown for water (Rumble, 2021) and hornworm plasma (Kenny et al., 2018). Data are means±s.d. The dashed lines represent the respective fit according to Arrhenius law. (B) Viscosity of bumblebee plasma in open geometry in the range 15–30°C as a function of waiting time (the inset shows a log-linear plot of the data). The constant viscosity values obtained in the sealed geometry for the same temperatures have been added (horizontal dashed coloured lines). Five experiments were performed per temperature, except at 15°C, where three experiments were performed. (C) Characteristic evaporation time te, defined for a viscosity of 1 Pa s (horizontal dashed black line in B), as a function of temperature. Evaporation time decreases with temperature, according to te=t0exp(−T/T1), where t0=144.9 min and T1=14.1°C (solid line).

Fig. 3B shows the evaporation dynamics of the bumblebee plasma – viscosity versus waiting time – between 15 and 30°C. We measured a significant increase in viscosity of 4–5 orders of magnitude over the duration of the experiment, typically 1 h. Evaporation, and thus viscosity increase, accelerates with temperature. At the end of each experiment, when the viscosity reached 104–105 Pa s, the material was so viscous that the Brownian motion of the beads could not be detected because it was below the resolution limit of the experimental device (camera and objective). A characteristic evaporation time te was defined for a viscosity of 1 Pa s, which corresponds to an increase of 3 orders of magnitude (1000 times the viscosity of water). We estimated that the evaporation time decreases with temperature, according to the empirical law te=t0exp(−T/T1), where t0=144.9 min and T1=14.1°C, as shown in Fig. 3C. The slow evaporation of plasma highlights the role of its constituents – proteins, amino acids – in the process. While a drop of pure water of the same volume would evaporate in about 10 min at 25°C, the drop of plasma showed an increased viscosity for more than 1 h while maintaining a large volume (typical humidity of 30%).

Conclusion

The aim of this study was to present a methodology for measuring the rheology of biological fluids available in small quantities, such as bumblebee plasma, using a particle tracking microrheology technique. We describe the impact of temperature on the rheology of plasma in a sealed geometry and show that the plasma viscosity follows an Arrhenius law at temperatures between 12.5 and 32.5°C. The activation energy is comparable to that of the hornworm larvae measured in a standard cone-plane viscometer, by Kenny et al. (2018), which requires relatively large samples of the order of 0.7 ml. Our rheological measurements with particle tracking microrheology show that plasma is a purely viscous fluid. This is consistent with the fact that it is composed of 90% water. It also contains numerous compounds such as amino acids and proteins (Wyatt, 1961; Chapman et al., 2013), which are insect dependent and are expected to have an impact on the activation energy. An interesting point here is that the difference between the activation energies of the insect plasmas is certainly related to the difference in their composition and could be considered a signature of the species in question. In the open geometry, evaporation of water in the plasma caused the viscosity to increase by 4– 5 orders of magnitude. Depending on the temperature, the time scale of evaporation ranged from tens of minutes to 1 h. These time scales are much longer than those that characterise coagulation, a mechanism that protects the insect from dehydration and pathogens by triggering a coagulation cascade that forms a seal. One might think that the slow evaporation of plasma maintains the integrity of the fluid that suspends the blood cells and proteins involved in clotting, allowing it to happen. Our experiments were carried out with 2–3 µl of sample, which is well suited to medium-sized insects. Fluid quantities in the tens of picolitres can also be considered, provided that the fluid of interest can be collected or extracted. These experiments pave the way for new measurements of viscosity in insects, for a more detailed characterisation.

We thank our colleagues Rudy Wattiez, Denis Michez and Corentin Decroo for fruitful discussions.

Author contributions

Conceptualization: A.L., J.B., B.A.; Methodology: A.L., B.M., V.T., O.S.-S., J.B., B.A.; Software: B.A.; Validation: A.L., B.M., V.T., O.S.-S., J.B., B.A.; Formal analysis: A.L.; Investigation: A.L., J.B., B.A.; Resources: B.A.; Data curation: A.L.; Writing - original draft: A.L., J.B., B.A., B.M.; Writing - review & editing: A.L., J.B., B.A.; Visualization: A.L., J.B., B.A.; Supervision: J.B., B.A.; Project administration: J.B., B.A.; Funding acquisition: J.B., B.A.

Funding

This project received funding from the Clean Sky 2 Joint Undertaking (JU) under grant agreement no. 864769. The JU receives support from the European Union's Horizon 2020 Research and Innovation program and the Clean Sky 2 JU members other than the Union. B.M. was funded by a postdoctoral fellowship from FRS-FNRS (Fonds de la Recherche Scientifique).

Data availability

All relevant data can be found within the article and its supplementary information.

Abou
,
B.
,
Gay
,
C.
,
Laurent
,
B.
,
Cardoso
,
O.
,
Voigt
,
D.
,
Peisker
,
H.
and
Gorb
,
S.
(
2010
).
Extensive collection of femtolitre pad secretion droplets in the beetle Leptinotarsa decemlineata allows nanolitre microrheology
.
J. R. Soc. Interface
7
,
1745
-
1752
.
Aprelev
,
P.
,
Bruce
,
T. F.
,
Beard
,
C. E.
,
Adler
,
P. H.
and
Kornev
,
K. G.
(
2019
).
Nucleation and formation of a primary clot in insect blood
.
Sci. Rep.
9
,
3451
.
Arafah
,
K.
,
Voisin
,
S. N.
,
Masson
,
V.
,
Alaux
,
C.
,
Le Conte
,
Y.
,
Bocquet
,
M.
and
Bulet
,
P.
(
2019
).
Maldi-MS profiling to address honey bee health status under bacterial challenge through computational modeling
.
Proteomics
19
,
1900268
.
Bidla
,
G.
,
Lindgren
,
M.
,
Theopold
,
U.
and
Dushay
,
M. S.
(
2005
).
Hemolymph coagulation and phenoloxidase in Drosophila larvae
.
Dev. Comp. Immunol.
29
,
669
-
679
.
Burdine
,
J. D.
and
McCluney
,
K. E.
(
2019
).
Differential sensitivity of bees to urbanization-driven changes in body temperature and water content
.
Sci. Rep.
9
,
1
-
10
.
Chapman
,
R. F.
,
Simpson
,
S. J.
and
Douglas
,
A. E.
(
2013
).
The Insects Structure and Function
, 5th edn.
Cambridge University Press
.
Chown
,
S. L.
,
Sørensen
,
J. G.
and
Terblanche
,
J. S.
(
2011
).
Water loss in insects: an environmental change perspective
.
J. Insect Physiol.
57
,
1070
-
1084
.
De Guzman
,
J.
(
1913
).
Relacion entre la fluidez y el calor de fusion
.
Anales de la Sociedad Espanola de Fisica y Quimica
11
,
353
-
362
.
Einstein
,
A.
(
1905
).
Uber die von der molekularkinetischen theorie der warme geforderte bewegung von in ruhenden flussigkeiten suspendierten teilchen
.
Ann. Phys.
322
,
549
-
560
.
Eyring
,
H.
(
1935
).
The activated complex in chemical reactions
.
J. Chem. Phys.
3
,
107
-
115
.
Eyring
,
H.
(
1936
).
Viscosity, plasticity, and diffusion as examples of absolute reaction rates
.
J. Chem. Phys.
4
,
283
-
291
.
Halsch
,
C. A.
,
Shapiro
,
A. M.
,
Fordyce
,
J. A.
,
Nice
,
C.
,
Thorne
,
J. H.
,
Waetjen
,
D. P.
and
Forister
,
M. L.
(
2021
).
Insects and recent climate change
.
Proc. Natl. Acad. Sci. USA
12
,
118
.
Heinrich
,
B.
(
1995
).
Insect thermoregulation
.
Endeavour
19
,
28
-
33
.
Heinrich
,
B
. (
2004
).
Bumblebee Economics
.
Harvard University Press
.
Hillyer
,
J. F.
and
Pass
,
G.
(
2020
).
The insect circulatory system: Structure, function, and evolution
.
Annu. Rev. Entomol.
65
,
121
-
143
.
Kanost
, (
2009
).
Hemolymph
. In
Encyclopedia of Insects
, 2nd edn.
Elsevier
.
Kenny
,
M.
,
Giarra
,
M. N.
,
Granata
,
E.
and
Socha
,
J. J.
(
2018
).
How temperature influences the viscosity of hornworm hemolymph
.
J. Exp. Biol.
221
,
jeb186338
.
Liebhold
,
A.
and
Bentz
,
B
. (
2011
).
Insect Disturbance and Climate Change
.
US Department of Agriculture, Forest Service, Climate Change Resource Center
.
Martinet
,
B.
,
Dellicour
,
S.
,
Ghisbain
,
G.
,
Przybyla
,
K.
,
Zambra
,
E.
,
Lecocq
,
T.
,
Boustani
,
M.
,
Baghirov
,
R.
,
Michez
,
D.
and
Rasmont
,
P.
(
2021
).
Global effects of extreme temperatures on wild bumblebees
.
Conserv. Biol.
35
,
1507
-
1518
.
Mason
,
T.
(
2000
).
Estimating the viscoelastic moduli of complex fluids using the generalized stokes-Einstein equation
.
Rheol. Acta
39
,
371
-
378
.
Menzel
,
F.
,
Morsbach
,
S.
,
Martens
,
J. H.
,
Räder
,
P.
,
Hadjaje
,
S.
,
Poizat
,
M.
and
Abou
,
B.
(
2019
).
Communication versus waterproofing: the physics of insect cuticular hydrocarbons
.
J. Exp. Biol.
222
,
jeb210807
.
Nation
,
J. L.
(
2016
).
Insect Physiology and Biochemistry
, 3rd edn.
CRC Press
.
Robinet
,
C.
and
Roques
,
A.
(
2010
).
Direct impacts of recent climate warming on insect populations
.
Integr. Zool.
5
,
132
-
142
.
Rumble
,
J
. (
2021
).
CRC Handbook of Chemistry and Physics
.
CRC Press
.
Siddiqui
,
M. I.
and
Al-Khalifa
,
M. S.
(
2014
).
Review of haemocyte count, response to chemicals, phagocytosis, encapsulation and metamorphosis in insects
.
Italian J. Zool.
81
,
2
-
15
.
Soroye
,
P.
,
Newbold
,
T.
and
Kerr
,
J.
(
2020
).
Climate change contributes to widespread declines among bumble bees across continents
.
Science
367
,
685
-
688
.
Sprenger
,
P.
,
Burkert
,
L. H.
,
Abou
,
B.
,
Federle
,
W.
and
Menzel
,
F.
(
2018
).
Coping with the climate: cuticular hydrocarbon acclimation of ants under constant and fluctuating conditions
.
J. Exp. Biol.
221
,
jeb171488
.
Tabunoki
,
H.
,
Dittmer
,
N. T.
,
Gorman
,
M. J.
and
Kanost
,
M. R.
(
2019
).
Development of a new method for collecting hemolymph and measuring phenoloxidase activity in Tribolium castaneum
.
BMC Res. Notes
12
,
2
-
7
.
Theopold
,
U.
,
Schmidt
,
O.
,
Söderhäll
,
K.
and
Dushay
,
M. S.
(
2004
).
Coagulation in arthropods: defence, wound closure and healing
.
Trends Immunol.
25
,
289
-
294
.
Waigh
,
T. A.
(
2005
).
Microrheology of complex fluids
.
Rep. Prog. Phys.
68
,
685
-
742
.
Waigh
,
T. A.
(
2016
).
Advances in the microrheology of complex fluids
.
Rep. Prog. Phys.
79
,
074601
.
Wyatt
,
G. R.
(
1961
).
The biochemistry of insect hemolymph
.
Annu. Rev. Entomol.
6
,
75
-
102
.

Competing interests

The authors declare no competing or financial interests.