Role of External Immune Stimulation and Its Impact on … of External Immune Stimulation and Its Impact on the Honey Bee Apis mellifera jementica in Saudi Arabia Hend M. Alharbi ,

Role of External Immune Stimulation and Its Impact on the Honey Bee Apis mellifera jementica Impact on the Honey Bee Apis mellifera jementica

in Saudi Arabia

Hend M. Alharbi , Ahmad Al Ghamdi , Ashraf M. Ahmed & presented by :Tahany H. Ayaad

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1- MrsHend M. AlharbiDepartment of Zoology, College of Science, King Saud University,2-ProfessorAhmad Al Khazim Al GhamdiChair of Engineer Abdullah Ahmad Bagshan for Bee Research, Bee

Research Unit, Plant Protection Department, Faculty of Food Sciences and Agriculture, King Saud University.Sciences and Agriculture, King Saud University.

3- ProfessorAshraf M. Ahmed Department of Zoology, College of Science, King Saud University

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�Honeybees are one of the well-known economic beneficial insects

known to man from time immemorial .

�In Saudi Arabia Beekeeping contributes to the traditional �In Saudi Arabia Beekeeping contributes to the traditional

economic development of the country by providing sustainable

additional income through self employment.

Antimicrobial

peptides

Honey

Pollen

BeewaxpollinationBeekeeping

IndustryIndustryBeewaxpollination

Propolis

Royal jelly

Bee venom

IndustryIndustry

Bees Hive ProductsBees Hive Products

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Fig. 1. The distribution of Apis mellifera jemenitica in Africa and Asia (all areas within

bold line) based on published reports (Ruttner, 1988; Hepburn & Radloff, 1998).

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� Apis mellifera jementica

(A.m.jementica) in KSA shows

high adaptation to the

environmental conditions in

the west, south and south westthe west, south and south west

regions during the hot summer

at temperature exceeds 45 °C.

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� Honey bee brood and adults areattacked by bacteria, viruses,protozoans, fungi and parasiticmites.

� The American Foulbrood(AFB) is an infectiousworldwide apiculturethreatening bacterial disease oflarvae and pupae transmittedby the Gram negativePanaebacillus larvae ( P.larvae).

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• Resistance ofP. larvae bacteria to

the currently used conventional

antibiotics [e.g. oxytetracycline,

Terramycin and tetracycline]

maximized the Apiculture threat by

this disease that impose urgentthis disease that impose urgent

demands for new classes of

therapeutic antibiotics.

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Research Objectives

� The current research is a ground step of a project entitled

“Humoral immune responses of the native honeybee, Apis

mellifera jementica towards developing natural antibiotics

against infectious food-borne diseases“

� This project is funded by the National Plan for Science and

Technology of King Saud University (project code numberTechnology of King Saud University (project code number

ASTP-10 ) .

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� Rsearch aims herein focuses oninduction of the humoral immuneresponse of workerA.m. jementica inresponse to injection oflipopolysaccharides (LPS) orMicrococus luteus (M.luteus)simulating the action of bacterialinfection.

� Bees are fed on standardized dose of Thimoquinone TQ for 72 hrs, then injected with LPS or M. luteus

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Thimoquinone Structure

2-Isopropyl-5-methyl-1,4-benzoquinone

� AMPs elicited in worker hemolymph treated groups are

evaluated in vitro by an agar well diffusion assay against

E. coli as Gram-negative bacteria or a panel of Gram

positive – bacteria.

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Examples of apiaries with local hives established in open fields or in constructions

Experimental groups (Gs)

Control G1

LPS

G2

M luteus

G3

LPS+TQ

M luteus+TQ

� An aliquot (0.2 ml) of a fresh`overnight culture wasspread onto agar plates (Ø59 cm) containing NB and LBmedium, respectively.

� As soon as the bacterial layer had been adsorbed, 1.5 ml ofundiluted hemolymph samples were applied as a dropletonto the plates with a pipette tip.

� After 24 h of incubation in an incubator at 37°C, thediameter of the clear zone of inhibition was measured anddocumentedby photography.documentedby photography.

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� Zone-inhibition assay for the detection of antimicrobial

activities in the hemolymph of infected bees were done.

� Aliquots of fresh overnight cultures of the Gram- negative or

Gram-positive bacteria (0.2ml) were spread on agar plates.

� Hemolymph aliquots (50 µl) directly applied onto the agar

plate and incubated overnight at 37°°C.

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plate and incubated overnight at 37°°C.

Results

Statistical AnalysisStatistical Analysis

�Results were statistically

analyzed using software (SPSS)

version number 12 (One-Way

Analysis of Variance ANOVA) and

the comparison between the

�Results were statistically

analyzed using software (SPSS)

version number 12 (One-Way

Analysis of Variance ANOVA) and

the comparison between the

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the comparison between the

groups is significant with high P

value ≤0.025 and insignificant if

the value P ≥ 0.025 .

the comparison between the

groups is significant with high P

value ≤0.025 and insignificant if

the value P ≥ 0.025 .

Zone Inhibition Assay for detection of anitmicrobial activity in hemolymph of LPS injected A.m. jementica towards E.coli

Control

1

1

TQ+ LPS

LPS

injection

1

control

LPS injected

TQ fed + injectionLPS

LPS

injection

TQ +LPS TQ +LPS injection

M.luteus B.subtilis St. aureus

Zone Inhibition Assay for detection of anitmicrobial activity in hemolymph of M.luteus injected A.m. jementica towards E. coli

bacteria

Control Control

1

1

TQ+ M.luteusTQ+ M.luteus

M.luteus

injection

M.luteus

injection

1

Zone Inhibition Assay for detection of anitmicrobial activity in hemolymph of M.luteus injected A.m.

jementica towards E. coli bacteria

Control

TQ+ M.Luteus

M.Luteus

Zone Inhibition Assay for detection of anitmicrobial activity in hemolymph of M.luteus injected A.m. jementica towards different

gram positive bacteria

M.Luteus

TQ+

M.luteus

M.luteus St. aureus B.subtilis

� The obtained data are in good agreement with Miyagietal. ( 2000) in which they reported that treatment of beeswith fungicides and antibiotics leads to the suppression ofthe bees’ immune system, the emergence of resistantpathogens, and the contamination of bee products .

� Enhancement of the honey bees immune systembyincreasing the expression level of AMPs in beesincreasing the expression level of AMPs in beesthemselves, is the solution to these problems (Bilikovaetal., 2001).

� Shen et al. (2010) have confirmed and chemicallysynthesized a proline rich peptide (PPO30) active towardsboth Gram negative and Grampositive bacteria

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Conclusion

� The current study could be used to build on approaches toenhance the immune systemof this native honeybee towithstand the extreme environment of KSA, as well asdeveloping protective therapeutic antibiotic(s) via therecombinant technology to control the most threateningrecombinant technology to control the most threateningbees food-borne diseases.

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AcknowledgmentAcknowledgmentAcknowledgmentAcknowledgment

The National Plan for Science and Technology of

King Saud University King Saud University (project code number ASTP-

10 ))

2010 - هـ 143128

Thank You

2010 - هـ 143129