susceptibility of red flour beetle life stages to elevated
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Susceptibility of Red Flour Beetle Susceptibility of Red Flour Beetle Life Stages to Elevated Life Stages to Elevated
TemperaturesTemperaturesRizana Mahroof, Kun Yan Zhu, and Rizana Mahroof, Kun Yan Zhu, and
Bhadriraju SubramanyamBhadriraju SubramanyamKansas State UniversityKansas State University
OverviewOverview• Introduction• Objectives• Methods• Results• Conclusions• Future work
What is the Rationale for Using Heat to What is the Rationale for Using Heat to Kill Insects?Kill Insects?
• Resistance in insects to chemicals, including fumigants
• Facility not suitable for fumigation• Companies do not want to use chemicals• Environmental problems associated with chemical
use
Thermal Death Effects of Heat on Thermal Death Effects of Heat on InsectsInsects
• Heat paralysis / anesthesia• Asphyxiation (production of excess CO2)• Coagulation of protoplasm• Coagulation of proteins • Destruction of enzymes essential to nerve
conduction • Decrease of hemolymph pH
– Desiccation and cellular abnormalities cause death
Susceptibility of Insects to Elevated Susceptibility of Insects to Elevated TemperaturesTemperatures
• Mortality depends on many factorsEnvironmental conditions (Temperature, RH)SpeciesStage and age of insectsTemperature historyHeat tolerance
How Heat Tolerance is Acquired ?How Heat Tolerance is Acquired ?• Thermal acclimation
• Through selection
• Synthesis of Stress Proteins/Heat Shock Proteins (HSP)Refolding of denatured proteins
Stabilizing proteins by removal of coagulated proteins
Folding and translocation of polypeptides
Assembly and disassembly of oligomeric protein complex
Roles in immune responses
HSP 73 is constitutive and HSP 72 stress-inducible
Objective 1Objective 1To identify the most heat tolerant stage of T. castaneum ?
Test InsectsTest InsectsTribolium castaneum (Herbst), Red Flour Beetle
(Tenebrionidae: Coleoptera)
Materials and MethodsMaterials and Methods
http://bru.gmprc.ksu.edu/ImageDB
• T. castaneum life stagesEggs (2-d-old)Young larvae (6-d-old)Old larvae (22-d-old)Pupae (26-d-old)Adults (2-wk-after emergence)
Life stages exposed to high temperatures
• 42, 46, 50, 54, 58 and 60oC• For varying lengths of time • 5 boxes containing 20 individuals
per box for each time interval
• Reared at 28oC and 65% RH
• Mortality recorded
Figure describing LT99 of T. castaneum life stages as a function of temperature
Temperature (oC)50 52 54 56 58 60
LT99
(min
utes
)
0
50
100
150
200
250
300
350
400
450
Eggs
Young larvae
Old larvaePupae
Adults
Symbols: observed dataSolid line: fitted data
Objective 2Objective 2Does heat shock protein (HSP 70) mediate heat tolerance
in T. castaneum?
Test InsectsTest InsectsT. castaneum
EggsYoung larvae Old larvae Pupae Adults
• Heat shock28 and 40oC (1 hour) and 23oC control
• Total protein concentrationBCA assay with kinetic microplate reader
Standard protein curve
Standard amount of protein loaded: 80 µg / well
Heat Shock Protein AnalysisHeat Shock Protein Analysis
Heat Shock Protein Analysis (Cont….)Heat Shock Protein Analysis (Cont….)• Western Blot Technique
4-20% Tris-glycine pre-cast gels (1.3% bis-acrylamide; Invitrogen)
Standard HSP - HSP 70 from bovine brain (Sigma)Molecular size marker – The MagicMarkTM Western Standard (Invitrogen)
Primary antibody – Monoclonal anti HSP 70 from mouse immunized with bovine brain HSP70 (clone BRM 22, Sigma)
Secondary antibody - Anti-mouse IgG conjugated with alkaline phosphatase (Invitrogen)
Immunodetection performed using the chemiluminescent method and the membrane exposed to X-Ray film for 2 mins.
Gray Value Quantification of Protein BandsGray Value Quantification of Protein BandsBands quantified using densitometric image analysis system (Ambis Imaging System and GelExpert Analysis System; Nucleotech Corporation).Absolute gray values quantified after background subtractionThe gray value of the 23oC samples (control) set arbitrarily to 100% as standard reference.The relative percentage of gray values for 28 and 40oC calculated based on standard reference.Data analyzed using Proc GLM and mean separation done using Fisher’s least significant difference (LSD).Results given as percentage relative HSP 70 values ± standard error
Temperature (oC)
HS
P 7
0 (%
)0
25
50
75
100
125
150
23 28 40
100.0a
83.1ab
65.5b
HSP Detected in Eggs Exposed to Different HSP Detected in Eggs Exposed to Different Temperatures Temperatures
Calculated molecular mass of HSP (70) is 75 kDa
F = 0.4.17; d.f. = 2; P = 0.05; n = 4 (Proc GLM, LSD)
MM HSP 23oC 28oC 40oC
75
120100806050
40
30
20
kDa
HSP Detected in Young Larvae Exposed to HSP Detected in Young Larvae Exposed to Different Temperatures (Larger MM Bands)Different Temperatures (Larger MM Bands)
kDa
MM HSP 23oC 28oC 40oC
120100
806050
40
30
20
24
69
Calculated molecular mass of HSP (70) is 75 kDa
Temperature (oC)H
SP
70
(%)
0
25
50
75
100
125
150
23 28 40
ba a
100.0129.0 133.4
F = 6.72; d.f. = 2; P = 0.01; n = 4 (Proc GLM, LSD)
Temperature (oC)
HS
P 7
0 (%
)0
255075
100125150175200225
23 28 40
a
a a
100.0
142.4.0 169.1
HSP Detected in Young Larvae Exposed to HSP Detected in Young Larvae Exposed to Different Temperatures (Smaller MM Bands)Different Temperatures (Smaller MM Bands)
kDa
MM HSP 23oC 28oC 40oC
120100
806050
40
30
20
24
69
Calculated molecular mass of HSP (70) is 75 kDa
F = 2.59; d.f. = 2; P = > 0.05; n = 4 (Proc GLM, LSD)
HSP Detected in Old Larvae Exposed to Different HSP Detected in Old Larvae Exposed to Different TemperaturesTemperatures
MM HSP 23oC 28oC 40oC
kDa
120100
80
605040
30
20
69
Calculated molecular mass of HSP (70) is 75 kDa
F = 0.62; d.f. = 2; P = > 0.05; n = 4 (Proc GLM, LSD)
Temperature (oC)
HS
P 7
0 (%
)0
25
50
75
100
125
150
23 28 40
a a a100.0 111.3 119.5
HSP Detected in Pupae Exposed to Different HSP Detected in Pupae Exposed to Different Temperatures Temperatures
MM HSP 23oC 28oC 40oC
kDa
12010080
6050
40
30
69
Calculated molecular mass of HSP (70) is 75 kDa
F = 2.57; d.f. = 2; P = > 0.05; n = 4 (Proc GLM, LSD)
Temperature (oC)
HS
P 7
0 (%
)0
25
50
75
100
125
150
23 28 40
a
a
a100.0
64.7
95.4
HSP Detected in Adults Exposed to Different HSP Detected in Adults Exposed to Different TemperaturesTemperatures
MM HSP 23oC 28oC 40oC
kDa
120100
80
6050
40
30
20
73
Calculated molecular mass of HSP (70) is 75 kDa
F = 0.76; d.f. = 2; P = > 0.05; n = 4 (Proc GLM, LSD)
Temperature (oC)
HS
P 7
0 (%
)0
25
50
75
100
125
150a
a a100.0
87.4 94.4
23 28 40
SummarySummary• Young larvae were the most heat tolerant stage while eggs were the least
heat tolerant.
• The expression of HSP 70 in young larvae was significantly increased by about 33% when temperature increased from 23 to 40oC. However, for eggs the expression of HSP 70 decreased by about 35%.
• For old larvae, pupae and adults the expression of HSP 70 did not vary significantly.
• Increased thermotolernace in young larvae could be due to either increased expression of HSP70 at higher temperatures and / or the additional HSP with lower molecular mass of 24 kDa.
• Reduced thermotolerance in eggs may be due to the denaturation of HSP70 with increasing temperatures.
Future ResearchFuture Research• To study the stability of HSP from young larvae exposed to 40oC for
different time periods.• To study the stability of HSP from young larvae exposed to 40-60oC.• Molecular characterization of different HSP in relation to different
temperature-time treatments.
AcknowledgementsAcknowledgementsTemp-Air, Burnsville, MN and CSREES-USDA (RAMP) Agreement No. 00-51101-9674
Dr. Brenda Oppert, GMPRC-USDA Manhattan, KS.