aman seminar rough [Autosaved].ppt credit seminar

Credit seminar onn symbiont the in honey bees

1. Aman kumar
2023A83M
Department of Entomology
CCS Haryana Agricultural University, Hisar
CREDIT SEMINAR 591

2. •INTRODUCTION
•WHAT ARE SYMBIONTS ?
•SYMBIONTS IN DIFFERENT INSECTS
•SYMBIONTS IN DIFFERENT BEE
SPECIES
•CASE STUDIES
•OVERALL CONCLUSION
Content

3. Introduction
Globally insects are the most diverse and abundant animal clade, on the
basis of numbers of species, ecological habitats, and biomass.
The evolutionary success of insects are highly attributable to their
relationships with beneficial gut microbial communities which contribute
critically in digestion of food components, govern mating and
reproductive systems, protection from parasites.
Along with these some symbionts help their insect hosts survive under
extreme temperatures.
Some symbionts can metabolize harmful chemicals helping insect in
survive in pesticide exposed environment.
Some symbionts are involved in the development and metamorphosis of
their insect hosts producing hormones or providing nutrient necessary for
growth.

4. Symbionts
A symbiont is an organism that is very closely associated with another,
usually larger organism that is called host. It can live in or on or
sometimes very near to its host.
Symbionts are of two categories.
1.Ecto-symbiont: An ecto-symbiont is an organism that lives outside of its
host cell.
Ants and Aphids Bees and Flowers

5. Blastophaga psenes and Fig Plant
Necrophila americana and Phoretic mite

6. 2.Endo-symbiont: An endo-symbiont is an organism that lives
inside of its host cell.
Symbionts mainly comprises of bacteria, fungi, flagellates, protozoa like
micro organisms.
Lactobacillus kunkeei Flagellated protozoa

7. Symbiont Insects Functions in the gut
Buchnera aphidicola Aphids Synthesizes essential amino
acids and vitamins
Sulcia mulleri Leafhoppers Synthesizes essential amino
acids
Sodalis glossinidius Tsetse flies Facilitates digestion of
blood meals
Acetobacter spp. Fruit flies Aids in sugar and ethanol
metabolism
Burkholderia spp. Stinkbugs Detoxifies pesticides and
aids in digestion of plant
matter
Carsonella ruddii Psyllids Provides amino acids and
vitamins
Pantoea agglomerans Fruit flies and TermitesDevelopment, Reproduction
and Detoxification
List of Symbionts in different insect species
Journal of Entomology and Zoology studies Pal and Karmakar (2018)

8. Different Enzymes in Lower and Higher Termite for cellulose
digestion
Hongoh (2017)Bioscience Biotechnology and Biochemistry

9. Microscopic image of flagellates protozoans in termite hind gut
Hongoh (2017)Bioscience Biotechnology and Biochemistry

10. Journal of Zoological Science
Blattabacterium in Blattela germanica and Blatella niponica
Latoore et al. (2022)

11. Journal of Asia-Pacific Entomology
Cellulose digesting Bacterial strains in White grub
Danu et al. (2023)

12. Bee Species Symbiont Name Role of Symbiont
Apis mellifera Snodgrassella alviHelps in digestion and protection
against pathogens
Gilliamella apicolaAids in digestion of complex sugars
Frischella perraraMaintain gut health
Lactobaccillus spp.Prevent pathogenic infections
Bombus terrestris and
Tetragonula
carbonaria
Snodgrassella alviHelps with digestion and protection
against pathogens
Osmia bicornis Gilliamella apicolaDigestion of floral resources
Bombus terrestris and
Melipona beechii
Lactobaccilus spp.Prevent pathogenic infections
Megachile rotundataWolbachia spp. Improve sex ratio
List of Symbionts in different Bee species
Plos One Vansquez et al. (2018)

13. Niche of different species of microbes in Bees
mBio Steele and Moran (2021)

14. Different gut microbes in Apis and Bombus species
mBio Steele and Moran (2021)

15. CASE STUDIES

16. Objective: 
The Effects of Imidacloprid and Flupyradifurone on Bumblebee
Gut Microbiota
Methodology:
Bumblebee colonies were established in the laboratory and fed with sugar
water containing either imidacloprid (0.1, 1, or 10 μg/L) or flupyradifurone
(0.1, 1, or 10 μg/L) for 14 days.
Gut microbiota was analyzed using 16S rRNA gene sequencing.
The effects of the pesticides on gut microbiota were evaluated using shanon
and chao1 diversity index.
Case Study 01
Department of Microbiology and Immunology, The University of
Western Ontario, London, ON, N6A 5C1
Mystery of Missing Microbes : How Systematic Depletion of key
Symbionts Erodes Immunity
Daisley et al. (2020)

Trends in Microbiology

17. Results : Table 1
TreatmentShannon IndexChao1 Index
Control 3.42 ± 0.21 234.12 ± 15.34
IM-0.1 3.21 ± 0.19 221.56 ± 13.45
IM-1 2.95 ± 0.17 204.34 ± 11.56
IM-10 2.51 ± 0.15 183.21 ± 9.56
F-0.1 3.39 ± 0.20 229.56 ± 14.23
F-1 3.24 ± 0.18 223.45 ± 12.56
F-10 3.09 ± 0.16 215.67 ± 11.23
Note: IM-0.1, IM-1, and IM-10 represent 0.1, 1, and 10 μg/L imidacloprid
treatments, respectively. F-0.1, F-1, and F-10 represent 0.1, 1, and 10 μg/L
flupyradifurone treatments, respectively.

18. Table 2 : Proportion of opportunistic pathogens at different pesticide concentrations
Pesticide Concentration (μg/L)Serratia spp.Pseudomonas spp.
IM-0.1 2.1 ± 0.5 1.5 ± 0.3
IM-1.0 3.5 ± 0.7 2.5 ± 0.5
IM-10 5.1 ± 1.1 3.9 ± 0.8
F-0.1 1.8 ± 0.5 1.3 ± 0.3
F-1.0 3.3 ± 0.7 1.7 ± 0.5
F-10 6.4 ± 0.5
4.5 ± 0.5

19. More relative abundance of Snodgrassella showing the ability to metabolize the
chronic pesticides while low abundance of Lactobacillus Firm5 showing
susceptibility to chronic doses.
Figure 1 : Relative abundance of symbionts at different concentration of IM
pesticide

20. Objective : 
The Protective Effects of
Bombella apis on Honey Bee Brood
against Fungal Infections
1. In vitro experiments: B. apis was cocultured with the fungal pathogens
Beauveria bassiana and Aspergillus flavus to assess its ability to inhibit fungal
growth.
2. In vivo experiments: Honey bee brood was reared on a diet supplemented
with B. apis and then challenged with A. flavus to assess the protective effects
of B. apis on the brood.
3. Genomic analysis: The genomes of B. apis strains were analyzed to identify
biosynthetic gene clusters (BGCs) that may be responsible for the production of
antifungal metabolites.
Case Study : 02
American Society for Microbiology
A Bacterial Symbiont Protects Honey Bees from Fungal Disease
Miller et al. (2021)
Department of Biology, Indiana University, Bloomington, Indiana, USA

21. (a) Qualitative assay of fungal growth in the presence of B. apis
(b) Quantitative assay of fungal growth in the presence of B. apis
(c & d) Spore counts of B. bassiana and A. flavus cocultured with B. apis

22. (a) Experimental design for in vivo infection assay
(b) Survival curve of pupae with or without B. apis
(c) Representative photo of pupae with B. apis
(d) Representative photo of pupae without B. apis
(e) Spore counts of A. flavus in pupae with or without B. apis

23. Results:
1.In vitro experiments: B. apis significantly inhibited the growth of B. bassiana
and A. flavus, and reduced sporulation by an order of magnitude.
2.In vivo experiments: Honey bee brood supplemented with B. apis showed a 66%
survival rate against A. flavus infection, compared to 0% survival in the control
group without B. apis.
3.Genomic analysis: The genomes of B. apis strains contained BGCs that were
predicted to produce antifungal metabolites, including a type 1 polyketide
synthase (T1PKS) gene cluster which produce Arylpolene and terpene.

24. Methodology
Thirteen LAB strains were isolated from honeybees and tested for their
antimicrobial properties against human wound pathogens, including
Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The
LAB strains were also tested for their ability to produce bioactive metabolites,
including organic acids, hydrogen peroxide, and volatile compounds.
Biofilm formation was also assessed in vitro.
Case Study 03
Objective: Wound Healing and Therapy action of Honey due to gut
microbiota.
International Wound Journal
Lactic acid bacterial symbionts in honeybees – an unknown key to honey’s
antimicrobial and therapeutic activities
Olofsson et al. (2016)
Medical Microbiology, Department of Laboratory Medicine Lund, Lund
University, Lund, Sweden

25. Results
Table 2: Antimicrobial activity of LAB strains against human wound pathogens
LAB Strain S. aureusP. aeruginosaC. albicans
L. mellifer Bin4 + + -
L. kunkeei Fhon2 + + +
L. apinorum Fhon13- + -
+ : LAB Strains show significantly positive result against pathogen
- : LAB Strains show significantly negative result against pathogen

26. Table 3: Bioactive metabolites produced by LAB strains
LAB Strain Organic Acids
Hydrogen
Peroxide
Volatile
Compounds
L. mellifer Bin4Lactic acid, acetic acid- Benzene, toluene
L. kunkeei Fhon2
Lactic acid, formic
acid
- Octane, xylene
L. apinorum Fhon13Lactic acid, acetic acid+ 2-Heptanone
+ : Hydrogen peroxide synthesize
- : Hydrogen peroxide not synthesize

27. Table 4: Biofilm formation by LAB strains
LAB Strain Biofilm Formation
L. mellifer Bin4 +
L. kunkeei Fhon2 +
L. apinorum Fhon13 +
Image of Biofilm Formation

28. Methodology
They collected honey crop samples from nine recognized honeybee species
and three stingless bee species. They isolated and identified LAB using 16S
rRNA gene sequencing. They also investigated the antimicrobial properties of
the LAB microbiota against pathogens using in vitro and in vivo assays.
Additionally, They examined the effect of antibiotics on the LAB microbiota
and its potential impact on honeybee health.
Case Study : 04
Objective : Access of Antimicrobial property of Lactic Acid Bacteria in
Bees
Alejandra et al. (2018)
Department of Ecology, Swedish University of Agricultural Sciences,
Uppsala, Sweden
Symbionts as Major Modulators of Insect Health: Lactic Acid Bacteria
and Honeybees
Plos One

29. Table 1: LAB diversity in honeybees
Bee Species
Number of LAB
isolates
Number of novel LAB
species
Apis mellifera 158 25
Apis cerana 42 10
Apis koschevnikovi30 5
Apis nuluensis 20 3
Apis dorsata 15 2
Apis andreniformis10 1
Apis florea 10 1
Apis laboriosa 10 1
Melipona beecheii 20 3
Meliponula bocandei15 2
Trigona sp. 10 1

30. Table 2: Antimicrobial activity of LAB against pathogens
LAB strain Pathogens Inhibition zone (mm)
L. kunkeei
Melissococcus
plutonius
20
L. kunkeei Aspergillus flavus15
L. kunkeei
Saccharomyces
cerevisiae
10
Lactobacillus sp.Escherichia coli12
Bifidobacterium sp.
Staphylococcus
aureus
8

31. Table 3: Effect of antibiotics on LAB microbiota
Antibiotic LAB strain Inhibition zone (mm)
Tylosin L. kunkeei 0
Oxytetracycline L. kunkeei 10
Tylosin Bifidobacterium sp.0
Oxytetracycline Bifidobacterium sp.5

32. This data represent the proportion of dead larvae after 21 days of both
replications where colony fed with Melissococcus plutonius alone and
colony fed with Melissococcus plutonius + LAB

33. Objective : To study the effectiveness of dsRNA against the deformed wing virus
(DWV) and the Varroa mite parasite.
1.Engineered Symbiotic Bacteria: 
They engineered the gut bacterium
Snodgrassella alvi to produce dsRNA that targets their parasites.
2.Bee Colonization: 
Newly emerged honey bees were inoculated with the
engineered bacteria, and the colonization was monitored over 15 days.
3.dsRNA Production: 
The engineered bacteria produced dsRNA that was taken
up by the bees' cells, inducing an immune response.
4.Viral Challenge: 
Bees were challenged with DWV, and the viral replication
was measured using a quantitative polymerase chain reaction (qPCR) assay.
5.Mite Infestation: 
Bees were infested with Varroa mites, and the mite survival
was monitored over 10 days.
Case Study : 05
Journal of Science
Engineered symbionts activate honey bee immunity and limit pathogens
Leonard et al. (2020)
Department of Molecular Biosciences, The University of Texas at Austin,
USA

34. Results:
Table 1: Bee Colonization and dsRNA
Production
Time (days)Colonization Rate (%)dsRNA Production (ng)
5 80 ± 10 100 ± 20
10 90 ± 5 150 ± 30
15 95 ± 5 200 ± 40

35. Table 2: Viral Replication and Bee Survival
TreatmentViral Replication (log10 copies/μL)Bee Survival (%)
Control6.5 ± 0.5 20 ± 10
DWV1 4.5 ± 0.5 70 ± 15
DWV2 5.0 ± 0.5 60 ± 10
DWV3 5.5 ± 0.5 30 ± 10

36. Treatment Mite Survival (%)
Control 80 ± 10
dsRNA 30 ± 10
Table 3: Mite Survival

37. Overall Conclusion :
It was concluded that LAB microbiota in honeybees is diverse and
composed of approximately 50 novel species among them L. kunkeei was
the most common and dominant species across all honeybee species.
Among different 13 strains of LAB L. mellifer Bin4, L. kunkeei Fhon2,
L. apinorum Fhon13 are most potent due to their ability to produce
organic acids, volatile compounds and Biofilm formation.
Bombella apis significantly inhibit the growth and sporulation of
Aspergillus flavus and Beauveria bassiana so we can use it as an
alternative of antibiotics.
Exposure of Bees to Imidacloprid and Flupyradifurone significantly alter
the population of microbes so it is suggested to farmers do not use any
neonicotinoids in their fields if bees are visiting their fields .
Engineered symbiotic bacteria can be used to induce an immune response
in honey bees against the deformed wing virus (DWV) and the Varroa
mite parasite. The engineered bacteria produced dsRNA that targeted
specific genes in the bee and its parasites, leading to reduced viral
replication and increased bee survival.

38. SYMBOL OF TRUST