Susceptibility of Red Flour Beetle Life Stages to Elevated Temperatures Rizana Mahroof, Kun Yan Zhu, and Bhadriraju Subramanyam Kansas State University.

Susceptibility of Red Flour Beetle Susceptibility of Red Flour Beetle Life Stages to Elevated Life Stages to Elevated

TemperaturesTemperatures

Rizana 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 factors Environmental conditions (Temperature, RH) Species Stage and age of insects Temperature history Heat 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 1

To 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 stages Eggs (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 larvae

Pupae

Adults

Symbols: observed dataSolid line: fitted data

Objective 2Objective 2 Does heat shock protein (HSP 70) mediate heat tolerance

in T. castaneum?

Test InsectsTest InsectsT. castaneum

Eggs Young larvae Old larvae Pupae Adults

• Heat shock 28 and 40oC (1 hour) and 23oC control

• Total protein concentration

BCA 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 Bands Bands quantified using densitometric image analysis system (Ambis

Imaging System and GelExpert Analysis System; Nucleotech Corporation).

Absolute gray values quantified after background subtraction The 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

120

100

806050

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

80

60

50

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

25

50

75

100

125

150

175

200

225

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

80

60

50

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

6050

40

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

60

50

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.