Download Appendix 1: Methods Species selection Species were selected to

Appendix 1: Methods
Species selection
Species were selected to cover a wide range of latitudinal range extents and positions. Studied taxa were: D.
bicostatus (Schaum, 1864) - n = 7, range extent = 2.5, range position = 41.75; D. depressicollis (Rosenhauer,
1856) - n = 5, range extent = 1, range position = 37.5; D. hispanicus (Rosenhauer, 1856) - n = 53, range extent =
8.5, range position = 39.75; D. moestus (Fairmaire, 1858) - n = 36, range extent = 1, range position = 42; D.
latus (Stephens, 1829) - n = 20, range extent = 29.5, range position = 53.75.
Immune assays
Encapsulation
The encapsulation assay was adapted from [2]. The tip of a fine sterile entomological micro-pin was used to
perforate the intersegmental membrane between the beetle’s second and third sternite under a dissecting
microscope. A nylon implant (diameter 0.06 mm, length 10 mm) was inserted into the wound with the interior
filament section occupying half the beetle’s body length, where it would be exposed to the circulating
haemolymph [2]. After 20 h the implant was removed and mounted on a microscope slide. Degree of
encapsulation was measured by viewing the implant under LED light using a Bresser USB microscope (Meade
Instruments Europe GmbH, Germany). Images of the implant were taken at 80 × magnification and the mean
pixel value calculated using Image J software (v. 1.48, National Institute of Health, USA). The image was then
split into channels and the red channel inverted with 'LUT fire' applied (shows bright areas in orange and dim
areas in blue) to give the best melanin band distinction against the background and therefore the most
accurate encapsulation value. The encapsulation value was then subtracted from a control value (the mean
value for part of the filament that had remained outside the beetle) to give a mean value for the encapsulation
response per individual [2,3].
Parasite load
Beetles were visually assessed for external parasites (e.g. ciliates and fungi) under a dissecting microscope,
before dissection to check for internal macro parasites. Parasite load was assessed as the presence or absence
of flukes, ciliates or eggs inside or outside the body cavity. Beetles were then crushed using the plunger in a
2.5 mL syringe at 1:14 ratio in PBS (1 mg to 14 µL) and centrifuged for 2 min at 10,500 gav.
Antimicrobial peptide (AMP) activity
The Gram-negative bacterium Escherichia coli Migula 1895 (K-12 strain EMG2, NCTC) and the Gram-positive
bacterium Arthrobacter globiformis Conn 1928 (NCIMB Ltd) were first grown on Mueller-Hinton agar (Oxoid) in
a Petri dish (9 cm in diameter) at 37 °C and 27 °C, respectively. Fifty millilitre conical flasks containing 5 mL
Mueller-Hinton broth were inoculated with one colony per flask. The bacteria were grown over 24 h in a
shaking waterbath at their optimal temperatures, and adjusted to 109.5 and 108 cells ml-1 respectively, using
sterile Mueller-Hinton broth. AMP activity was measured using the zone of inhibition assay adapted from [4].
One hundred microliters of bacterial suspension at the appropriate cell densities were added to 10 mL of
sterile Mueller-Hinton agar at 48 °C, and poured into a sterile 9 cm Petri dish. The dish was gently swirled to
create a thin layer of agar and ensure even dispersal of the bacteria. Once set, eight evenly spaced 2 mm wide
wells were created in the agar and 3 µL of beetle extract was added in triplicate. As a negative control one well
contained 3 µL sterile phosphate buffered saline and as a positive control another well contained 3 µL
tetracyclin per plate (E.coli 0.0075 mg mL-1 and A. globiformis 0.125 mg mL-1). Plates were sealed with
Parafilm® and incubated over 96 h, at optimal temperature until a bacterial lawn was visible. The number of
clear zones produced was recorded and diameter measured using callipers at the widest and narrowest points.
The mean clear zone diameter was taken per sample and expressed as a percentage of the clear zone
produced by the positive control, to account for any differences between plates such as temperature variation.
Phenoloxidase (PO) activity
PO activity was measured corresponding to two points in the melanin synthesis pathway, dopachrome
production and consumption. For both reactions, 3 µL beetle extract was added to a 96-well ELISA plate
(Corning, NY) with each well containing 177 µL of 10 mM MOPS (containing 20 mM calcium chloride; pH 7) and
20 µL of 4.94 mg mL-1 L-dopa made up in the same MOPS buffer (for dopachrome production assay) and 20 µL
of dopachrome (10 µL of 2.5 mM L-dopa and 10 µL of 5 mM sodium periodate both made up in MOPS buffer
and mixed at a 1:1 ratio; for dopachrome consumption assay). Blanks contained 180 µL of MOPS buffer and 20
µL of L-dopa or L-dopachrome, respectively. A VersaMax® Pro microplate reader and SoftMax® Pro v.5.4
software (Molecular Devices Ltd, Berkshire, UK) was used to measure absorbance at 475 nm (for dopachrome
production) and 375, 475 and 575 nm (for dopachrome consumption) every min for 1 h. For dopachrome
production results, samples were blank corrected and for dopachrome consumption, average absorbance at
375 and 575 nm were subtracted from the values at 475 nm to reduce interference from other components
formed. Enzyme activity was measured as the slope of the reaction curve during the linear phase of the
reaction [5,6]. These values were then normalised against protein to aid in comparison between species.
Thermal ramping
To take into account the fact that species differ in their thermal windows a range of five environmentally
relevant sub-lethal temperatures were used to induce thermal stress. Individuals were transferred into 1 L
snap-lock plastic containers containing aerated artificial pond water (pH 7.7, acidified using hydrochloric acid
(ASTM, 1980) with 2 cm head space and 15 °C), and inert plastic vegetation for the beetles to hold on to whilst
submerged. The plants were added as we found that the beetles became increasingly stressed without them,
swimming frenetically and trying to climb out the water. The tanks were then placed in a covered polystyrene
water bath and submerged to lid level. The tanks were then maintained at 15 °C for 30 min prior to ramping to
allow the beetles to settle after being moved.
The animals were then maintained at 15 °C or ramped from 15 °C to 20, 25, 30 or 35 °C, at a rate of 0.25 °C
min-1 and held at temperature for 24 h using a refrigerated circulating bath with a heating circulator attached
(type R5 and GP200 respectively, Grant Instruments Ltd., Shepreth, UK) and Labwise software version 1, with
thermocouples monitoring temperature both submerged inside the water bath and inside the tanks.
Temperatures chosen were within the range experienced by Deronectes adults in the field and below the
upper thermal limits of all species. This ramping rate was chosen to mimic the warming of a shallow stream or
temporary pond in summer, and to ensure that any metabolic stress measured was due to the final
temperature held at and not the rate of thermal ramping.
Water quality was tested immediately before and after thermal ramping to ensure treatment temperatures
met and to monitor dissolved oxygen levels. Pre-ramping, temperature averaged 15.4 ± 0.1 °C, pH 8.1 ± 0.00,
salinity 0.1 ± 0.00 ppt, nitrates 0.0 ± 0.0 mg L-1 and dissolved oxygen 9.8 ± 0.4 mg L-1 and 98.8 ± 3.6% (see Table
1 for post-ramping). After ramping, animals were frozen in liquid nitrogen and crushed using the plunger of a
2.5 mL syringe at a 1:14 ratio either in cold 2.5% TCA and centrifuged before being neutralised using 9 µL of
3M sodium carbonate to every 0.1 mL of supernatant for ATP, lactate and succinate assays, or extraction
buffer (containing 100 mM potassium phosphate, pH 7.4, 1 mM DTT and 1 mM EDTA) for energy storage
molecule assays).
Table S1. Mean physiochemical parameters measured post-thermal ramping.
Temperature treatment (°C)
15
20
25
30
35
Temperature (°C)
14.960 ± 0.103
21.089 ± 0.257
24.78 ± 0.236
28.444 ± 0.106
33.222 ± 0.189
pH
7.733 ± 0.050
7.519 ± 0.050
7.416 ± 0.090
7.431 ± 0.037
7.4325 ± 0.024
77.900 ± 7.644
81.500 ± 1.880
78.360 ± 2.396
66.950 ± 7.193
66.189 ± 2.432
7.908 ± 0.158
6.557 ± 0.134
6.034 ± 0.166
5.252 ± 0.170
4.378 ± 0.143
DO (%)
-1
DO (mg L )
Means and standard error reported for temperature, pH and dissolved oxygen (DO) after 24 h.
Metabolic assays
ATP concentration
ATP production was measured as the light emitted in Relative Luminesence Units (RLU) over time using an ATP
Sustained Luminescence (SL) Kit (BioThema AB, Handen, Sweden) and a Pi-102 tube luminometer (Hygeina
International Ltd, Watford, UK) [7]. ATP was measured by adding 2 µL of sample to 148 µL Tris-acetate buffer
and 20 µL of ATP SL reagent in new glass luminometer tubes. Light emission was measured using the tube
luminometer every 15 s for 90 s [7]. After 105 s an internal standard of 2 µL 100 µM ATP made up in potassium
HEPES buffer (pH 7.5) was added and RLU measured again at 120 s. The ATP concentration per sample was
calculated using the following equation: ATPsmp = 10-7 x Ismp/(I(smp+std) - Ismp). The factor 10-7 is the molar
concentration of ATP standard in the cuvette (10 µl 10-5 M in a total reaction volume of 1 mL).
Lactate concentration
To measure lactate, 268 µL 0.4 M hydrazine - 0.5 M glycine buffer (pH 9 using 3 M hydrazine sulphate) was
added to 1 µL L-LDH from Rabbit muscle (Sigma-Aldrich, 25 µL 80 mM NAD+ and 6 µL sample in a two-step
addition. The glycine-hydrazine buffer was used as the hydrazine reacts with pyruvate, allowing for
quantitative conversion of lactate to pyruvate and hence the production of 1 mol of NADH per mol of lactate in
the sample. After 1 h incubation at 37 °C, the plate was transferred into a VersaMax® Pro microplate reader
and read using SoftMax® Pro v.5.4 software (Molecular Devices Ltd, Berkshire, UK) at 340 and 400 nm. This
method of measuring lactate concentration was amended from [8]. The absorbance at 400 nm was subtracted
from the measurement at 340 nm and samples subtracted from the blank to remove background noise from
the reading. The original concentration of NADH in the well was calculated using a standard curve and the
lactate concentration in µL mg-1 beetle calculated.
Succinate concentration
Succinate concentrations were measured using a Succinic Acid Kit (Megazyme International Ireland, Co.
Wicklow, Ireland). The spectrophotometer was zeroed on 67 μL buffer and 667 μL distilled water in a 1 mL UVcuvette. To run samples 67 μL NADH, 67 μL ATP/PEP/CoA solution and 2.4 μL neutralised extract was used with
6.7 μL pyruvate kinase/L-LDH added at 100 s, and 6.7 μL succinyl CoA synthetase added at 300 s. A kinetic
assay was ran over 10 min in a Jenway 7315 spectrophotometer (Bibby Scientific Ltd, Staffordshire, UK) at 340
nm. The sample succinate concentrations were below detectable limits.
Protein content
Protein, soluble carbohydrate (e.g. glucose), insoluble carbohydrate (e.g. glycogen) and total lipid content
were measured using procedures adapted from [9]. The benefit of using this step-wise procedure is that all of
the components mentioned can be measured in one individual, reducing the number of individuals required.
Protein was measured in accordance with the manufacturer's instructions in the micro-BCA® Protein Assay Kit.
Supernatant (2.5 µL) was transferred into a 96-well microplate being careful not to disturb the pellet or surface
lipid bilayer, together with 247.5 µL of BCA reagent (made up of BCA reagent A and B at a ratio of 50:1).
Samples were covered with PCR film and incubated at 37 °C for 30 min before being read at 562 nm. Blanks
were run of extraction buffer (100 mM potassium phosphate, 1 mM EDTA and 1 mM DTT, pH 7.4), and bovine
serum albumin standards made in extraction buffer. Blank absorbance readings were then subtracted from
sample readings.
Glucose concentration
To extract glucose, glycogen and lipids from the same insect homogenate, carbohydrates were dissolved using
11.1 µL of 20% sodium sulphate solution to every 100 µL sample to reach a final solution of 0.2 mL of 2%
Na2SO4 and centrifuged to precipitate the glycogen. The supernatant was then mixed with 833.3 µL of 100%
chloroform - 80% methanol solution (1:2 v/v) 100 µL-1 supernatant to separate the total lipids from the glucose.
The supernatant was then vortexed vigorously, before centrifugation for 15 min at 180 gav and 4 °C. The
supernatant containing free sugar in the upper phase and lipids in the lower phase was transferred into a fresh
Eppendorf and the glycogen pellet kept for determination of glycogen content.
Glucose and glycogen were determined using a colorimetric method based on anthrone reagent in a two-step
procedure. Supernatant (100 µL) was transferred in to snap-lock Eppendorfs and the chloroform and methanol
evaporated off at 85 °C for 1 h until ~10 µL were left using a Gallenkamp size 2 incubator (Gallenkamp and
Company Ltd, London, UK) in a vented fume hood with a chemcap® multipurpose filter (Bigneat Ltd,
Hampshire, UK). Anthrone reagent (240 µL of 1.42 g L-1 anthrone in 70% sulfuric acid) was added and the tubes
incubated at room temperature for 15 min, before heating at 90 °C for 20 min. The tubes were then cooled on
ice and vortexed gently by pulse mixing. Two hundred and twenty microliters was then transferred in to a flatbottom 96-well plate and the absorbance read at 625 nm on a VersaMax® Pro microplate reader using
SoftMax® Pro v.5.4 software (Molecular Devices Ltd, Berkshire, UK). Extraction buffer was used for the blank
and D-glucose standards of 0 - 1 mg mL-1 were made up in extraction buffer [9].
Glycogen concentration
Pellets containing sodium sulfate and bound glycogen were washed twice using 400 µL of 80% methanol,
before being vigorously vortexed and centrifuged for 5 min at 10,500 gav and 4 °C. The supernatant was then
carefully pipetting off and 555 µL 1.42 g L-1 anthrone reagent added 100 µL-1 of supernatant. This was heated
for 25 min using an incubator inside a ducted fume hood at 85 °C, before removing from the incubator and
cooling on ice to stop the reaction. The supernatant was vortexed and then pulse centrifuged to 6150 gav.
Glycogen content of 250 µL supernatant was then determined at 625 nm in triplicate with D-glucose as the
standard on a VersaMax® Pro microplate reader using SoftMax® Pro v.5.4 software (Molecular Devices Ltd,
Berkshire, UK). The assay is linear between 0 and 5 mg mL-1 D-glucose made up in 100 mM potassium
phosphate, 1 mM DTT and 1 mM EDTA.
Lipid concentration
The total amount of lipids in each insect was determined using the vanillin assay [9], a high throughput
technique providing accurate data for the determination of lipids in insects [10]. Vanillin reagent was prepared
by mixing vanillin with 70% orthophosphoric acid, reaching a final concentration of 1.2 g L -1 and stored safe
from light. Supernatant (75 µL) was added to polypropylene tubes in duplicate and the solvent evaporated off
in an incubator inside a ducted fume hood at 80 °C for 30 min for standards and 1 h for samples. Ten
microliters of 98% of sulphuric acid were added being careful to pipette this to the bottom of the tube,
vortexed gently to mix and heated for 3 min 80 °C. The samples were then removed from the incubator,
cooled on ice and vortexed gently to mix. Supernatant (5 µL) was then transferred into a fresh Eppendorf and
195 µL vanillin-orthophosphoric reagent added per well. The samples were then incubated at room
temperature for 15 min before transferring 180 µL per well to a 96 well plate. The absorbance was measured
spectrophotometrically at 525 nm using triolein diluted in 100% chloroform as standards. To calculate the
plasticity of each metabolic variable in response to temperature, the smallest value was subtracted from the
highest value, irrespective of which temperature those values were measured.
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Appendix 2: Results
Table S2. Principal component (PC) loadings for immune and metabolic variables in Deronectes.
Immune
variables
Metabolic
variables
metPC1
metPC2
-0.261
0.602
0.308
0.400
-0.438
-0.001
0.284
0.205
ΔATP
Δlactate
Δprotein
Δglucose
Δglycogen
Δlipids
-0.472
0.472
0.491
0.342
0.407
0.176
-0.046
0.310
0.004
-0.453
-0.362
0.752
-0.063
-0.441
-0.511
-0.631
0.252
-0.042
0.043
-0.275
ΔATP
Δglucose
Δlipids
0.250
-0.395
0.385
0.526
0.282
-0.542
immPC1
immPC2
Clear zone no. A. globiformis
Clear zone no. E. coli
Clear zone Ø A. globiformis
Clear zone Ø E.coli
Dopachrome production
Dopachrome consumption
Encapsulation ability
External parasite load
-0.437
0.119
-0.359
-0.402
0.195
-0.452
0.306
0.411
Independent contrasts
Clear zone no. A. globiformis
Clear zone no. E. coli
Clear zone Ø A. globiformis
Clear zone Ø E.coli
Dopachrome production
Dopachrome consumption
Encapsulation ability
External parasite load
-0.519
0.079
0.006
-0.005
-0.477
0.364
0.419
-0.435
For immune variables, antimicrobial peptide activity against Gram-positive bacterium Arthrobacter globiformis and
Gram-negative bacterium Escherichia coli, measured as the number and size of clear zones, phenoloxidase activity
measured as dopachrome production and consumption rates, and external parasite load were measured. For
metabolic PCs the plasticities of ATP, L-lactate, protein, glucose, glycogen and lipid were measured. PC loadings
explained 41-59% variation for PC1 and 25-31% variation for PC2. Parameters deemed significant are highlighted in
bold (> 0.50 or < -0.50).
Figure S1. Biplots for the most significant variables and principal component loadings predicting latitudinal
range extent and central position. Codes: Deronectes bicostatus (BIC), D. depressicollis (DEP), D. hispanicus
(HIS), D. moestus (MOE) and D. latus (LAT).
Appendix 3: Data
Table S3: Metabolic plasticity in Deronectes species studied. ATP, lactate, protein, glucose, glycogen and lipid plasticity were measured in response to thermal stress.
Species
ΔATP
Δlactate
Δprotein
Δglucose
Δglycogen
Δlipids
bicostatus
0.197
13.969
26.607
2.864
36.898
0.714
depressicollis
0.252
11.971
13.933
1.811
13.065
2.340
hispanicus
5.552
7.402
11.724
1.360
8.126
0.916
latus
moestus
0.815
1.648
4.720
5.347
10.503
5.280
1.761
2.465
28.199
7.561
0.163
0.000
Table S4: Immunocompetence in Deronectes species studied. Measures of immunocompetence included encapsulation ability (encap), antimicrobial peptide activity
against Gram-positive bacterium Arthrobacter globiformis and Gram-negative bacterium Escherichia coli i.e. the number (aglob_no and ecoli_no) and size (aglob and ecoli)
of clear zones, phenoloxidase activity (dopachrome production and consumption rates; dcprod and dccon) and external parasite load (out) of Deronectes species studied.
+1 = data had 1 added to raw data.
Encapsulation
ability
Clear zone no. A.
globiformis +1
bicostatus
26.700
42.857
depressicollis
16.558
hispanicus
latus
moestus
Species
Clear zone no.
E. coli +1
Clear zone Ø
A. globiformis
Clear zone Ø
E.coli +1
Dopachrome
production +1
Dopachrome
consumption
External parasite
load +1
29.571
7.988
1.000
1.006
0.132
86.714
66.667
11.000
9.116
1.000
58.657
0.082
81.000
17.069
65.790
9.571
15.011
8.727
1.180
0.659
40.024
22.111
19.466
72.222
76.667
6.263
41.000
6.500
13.363
1.000
32.484
1.365
1.131
0.620
0.301
1.000
43.216
Table S5: Thermal performance, dispersal ability (DA) and body mass (BM) of Deronectes species. ΔUTL = acclimatory ability of upper thermal tolerance and ΔLTL =
acclimatory ability of lower thermal tolerance.
Species
ΔUTL
ΔLTL
Absolute thermal
tolerance range
Dispersal
ability
Body
mass
bicostatus
-0.35
0.762
53.9
1.15
6.82
depressicollis
0.72
0.606
52.89
1.11
6.28
hispanicus
1.347
-0.529
50.72
1.2
9.88
latus
moestus
2.023
0.173
0.459
1.087
56.87
54.44
1.01
1.13
7.96
6.01