PPT explains how Higher termites evoled, how termites acquired gut microbes, How they lost gut protists

1. Welcome
1

2. Termite evolution
Rise of Termitidae
2
Kishor Pujar
PALB 9014
II Ph.D.
Agricultural Entomology
University of Agricultural Sciences Bangalore

3. 3
When did termites originated?
How did termites acquired the gut
symbionts?
How eusociality developed within
them?
How these termites lost their gut
protozoa?

4. 4
Blattoidea
Corydoidea
(Polyphagoidea)
Blaberoidea
Blattoidae
(Cockroaches)
Blattidae
Oriental cockroach
Blatta orientalis
Blaberidae
Giant Cockroach
Blaberus giganteus
Ectobidae (Blattelllidae)
German cockroch,
Blattella germanica
Termitoidae
(Termites)
Cryptocercidae
(Wood roches)
Cryptocercus darwinii
Blattodea
Corydiidae
(Sand Cockroaches)
Polyphaga pellucida
Closely related

5. Proposed epifamily classification shown on the tree topology from Inward et al. (2007)
Termites
Super order: Dictyoptera
Order: Blattodea
Infra order: Isoptera
Epifamily: Termitoidae
5
Cockroch
Mantid
Termites

6. 6
Mastotermes darwiniens
Hodotermitidae
Mastotermitidae
Archotermopsidae
Stolotermitidae Kalotermitidae
Serritermitidae
Stylotermitidae
Rhinotermitidae Termitidae
Basal groups Neoisoptera
Higher termites

7. 295 species, 52
genera, and six
families
Hodotermitidae,
Kalotermitidae,
Rhinotermitidae,
Stylotermitidae and
Termitidae
59 species (42.75%)
reported are endemic
5 genera are endemic
with eight species.
Southern Indian
132 species 37 genera
from five families
7
Termites
3176 known
species
2976 living
200 fossil (Constantino, 2020)
Termitinae
19 species out of
42 are endemic
Hodotermitidae: Anacanthotermes viarum Stylotermitidae: Stylotermes fletcheri
Termites (Blattodea: Isoptera) of southern India: current knowledge on distribution
and systematic checklist, M. Ranjith & C.M. Kalleshwaraswamy. 26 May 2021

8. 8
Highlights of Termitidae
150 million
years ago…………
75% of termites
True workers
Unique gut morphology and
physiology
Different microbial community
Various feeding habit

9. 9
Mesozoic
Triassic
245-210 MYA
Jurassic
210-145 MYA
Creataceous
145-65 MYA
Cenozoic Tertiary
Palaeocene
65-60 MYA
Eocene
60-35 MYA
Oligocene
35-25
MYA
Meocene
25-5 MYA
Pliocene
5-1.5 MYA
Termites
140 MYA
Blattodea (Cockroaches)
350 MYA
Palaeozoic
Cambrian
600-500
MYA
Ordovician
500-440
MYA
Silurian
440-400
MYA
Devonian
400-360
MYA
Carboniferous
360-285 MYA
Permian
285-245
MYA
Mantodea
60 MYA
Era
Era
Era
Periods
Periods
Period
Epochs
Insects
Geological time scale of termites
Vespid wasp
118 Ma,
Formicoidea
140-150 Ma

10. 10
Lebanese amber - 130-125 million years
(Early cretaceous)
Lebanotermes veltzae n. gen., n. sp., holotype
dorsal view
Cosmotermes opacus soldier
Burmese amber (98 -100 million years old (Mid cretaceous)
C. multus paratype soldier C. opacus sp. nov.,
Cambay amber (Early Eocene) 50 and 52 million years old
A. Nanotermes isaacae
Engel & Grimaldi,
gen. et sp. n.,
holotype ,
B. Parastylotermes
krishnai Engel &
Grimaldi, sp. n.,
holotype,
C. Zophotermes
ashoki Engel &
Singh, sp. n.,
holotype
Termitidae: Tad-262
Stylotermitidae:Tad-277
Rhinotermitidae: Tad–42
Dominican amber 25-40 million years old (Oligocene to Miocene)
Holotype imago of
Archeorhinotermes rossi, n. sp
Anoplotermes carib, n. sp
Ambers

11. 11
Species Time Place
Meiatermes bertrani
(Hodotermitidae;
Hodotermitinae)
lithographic limestone dating to ~130
Ma
Montsec, Lleida, Spain
M. araripena Santana limestone 110 Ma Brazil (Aptian to lower Aptian,)
Valditermes brenanae,
Mastotermitidae
Weald Clay of Surrey -120 Ma England (Hauterivian )
Most modern species of termites are tropical, most species of fossil Hodotermitidae
are temperate or warm-temperate
Fossils of the genus Meiatermes found in lower Cretaceous deposits in Brazil and
even older limestone in Spain
Gondwanan distribution Land bridges Storms
Distribution

12. 12
The Evolutionary History of Termites as Inferred from 66 Mitochondrial Genomes
Sequenced the mitochondrial genome of 66 termite
species (48 sequences from this study + 18 from GenBank
Nontermite taxa included in this analysis were
Periplaneta fuliginosa, Cryptocercus relictus, Tamolanica
tamolana, Megacrania alpheus Locusta migratoria
Collection
Dissection
DNA extraction
Phylogenetic analysis by MEGA 5.2
Bayesian analysis and Maximum
likelihood method
Molecular dating by BEAST 1.8.0
To study the origins and diversification of termites
(Bourguignon et al., 2014)
Removed the digestive tract to avoid contaminants
from the gut (symbionts and soil bacteria, food
particles, soil minerals, etc.)
TaKaRa DNA kit, from five to ten individual specimens
Periplaneta fuliginosa, Blattella germanica,
Eupolyphaga sinensis, and Cryptocercus relictus; amantis,
Tamolanica tamolana; a Mantophasmatodea, Sclerophasma
paresiense; a phasmid, Megacrania alpheus; and the locust,
Locusta migratoria

13. 13
Species Time (MYA)
Cryptocercus roaches 170
Mastotermitidae 150
Hodotermitidae 130-140
Archotermopsidae 130-140
Kalotermitidae 125
Rhinotermitidae 90-100
Serritermitidae 70
Termitidae 54
Origin of ants
Diversification of angiosperms
Pangaea beak up- 200-80 MYA
Gondawana beak up- 130-80 MYA

14. 14
Blaberus 6-7 cm fecal pellet
(Carboniferous period)
Devonian and continuing through the Carboniferous,
primary production was overwhelmingly routed through
detritivores
Parenchyma in pellet
Diversification of angiosperms resulted in shift from coprophagous to xylophagous
Ancestor of
wood roach
and termites
Detrivorous Coprophagy + Xylophagous
Food
,`,
`,`
`
,````$
$`,;`,’.!!
!!.’.....’’,
…,…``
0 00 0
00 0 0
0 0 0
0 0 0
0
``` 0
0 0
0 00 0
00 0 0 0
0 0 0 0
0
0
```
00```
`
0 0
0 ```
00`
FC
C
Coprolite

15. 15
Protists
Flagellated Cliated Sporozoans Amoeboid
Protozoa
Metamonada
Parabasalia
Hypermastigida
Trichonympha
Class
Order
Genus
Lower Termites
and
wood roaches
Phylum
Trichonympha

16. 16
Cryptocercus punctulatus Hodotermopsis sjoestedti Reticulitermes speratus
Cp20 Cp07 Cp13 Cp49 Cp38 Cp26
Dissection of
gut
DNA
extraction
SSU rRNA
gene
pCR2.1-TOPO
vector
66 clones
sequenced
Selected
representative
sequence
FISH for the
identification
Phylogenetic
analysis
PHYML
software
Maximum
likelihood
mehod
Bootstrap
100 replicates
(Ohkuma et al.,2009)
Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the
cockroach Cryptocercus

17. 17
T. acuta- Cp20 Cp07
Trichonympha sp.
Cp13
Urinympha talea
Cp49
Barbulanympha ufalula
Cp38
Barbulanympha sp
Cp26
Barbulanympha sp.

18. 18
The Cryptocercus and termites acquired gut protists from their common ancestor
Cryptocercus and termites harbours the similar flagellates (Trichonympha spp.)
Conclusion
Inference
Trichonympha
Urinympha talea
Barbulanympha
Eucomonympha imla
Trichonympha
Hoplonympha
Eucomonympha species
Teranympha
Pseudotrichonympha
Cryptocercus Termites
(Yamin,1979)

19. 19
Subsociality induced proctodeal trophalaxis in ancestor
Protozoans
thrown out
through moulting
and faecus
Encystment of
protozoa
Only chance of
pass to hatchlings
by coprophagy
Reproduce in the
host
(SEELINGER and SEELINGER,1983)
Ancestor was
sub social and
semelparous
Adults stops
moulting
Protozoa pass
to hatchlings
by gut fluid
Proctodeal
trophallaxis
Acquisition of gut protists and their transmission through proctodeal
trophallaxis is inherited by the ancestor

20. 20
Proctodeal trophalaxis forced to alloparental care and eusociality in termites
Proctodeal
tropholaxis
Altricial
development
Biparental to
alloparental
care
Reproductives
relaxed from the
brood caring
Semelparity to
Iteroparity
Fecundity
increased
Eusociality
developed
Emergence of sterile
castes
Workers and Soldiers
Alloparental care has become irreversible

21. 21
Irreversible transfer of brood care duties and insights into the burden of caregiving in
incipient subterranean termite colonies
(Chouvenc and Yaosu, 2017)
Coptotermes gestroi (Wasmann)
Rhinotermitidae
After the brood care duty is transferred to alloparents, do the primary
reproductive become irreversibly dependent on alloparents
Do the survival, development, and growth of the brood rely on the nurturing
capacity of the group (i.e. the maximum amount of brood care that alloparents
can provide?
Collection
Moist
corrugated
cardboard
Identification
Rearing unit
One male and
female
dealate
250 colonies
were initiated

22. 22
.
A perforated plastic cap was placed on the top to allow for aeration, but to limit desiccation
and prevent escape. Units were kept at 28 ∘C for 150 days (∼5 months).
transparent plastic cylindrical vial (8 cm × 2.5 cm diameter, internal volume = 37 cm3)
with 6 g of moistened organic soil at the bottom (commercial organic potting soil) .
Four blocks of Picea sp. (5 cm × 0.5 cm × 0.5 cm) were positioned vertically and an additional
Picea block (10 cm × 0.5 cm × 0.5 cm) was placed inside the vial, along the vertical side.
3% agar solution was poured, leaving a 2-cm space at the top of the vial. When the agar was
solidified, the long Picea block was removed from the vial to leave a hole in the agar,
providing direct access to the soil on the bottom and to the wood.
Treatment Details
1 Zero workers all workers and soldiers were removed
2 One worker only one worker was left in the colony
3 2 workers only two workers were left in the colony
4 5 workers only five workers were left in the colony
5 10 workers only 10 workers and one soldier were left in the colony
6 All workers all workers (35) and soldiers (four) were left in the colony
At 150 days, more than 50 live
colonies opened
28.1 ± eggs,
8.4 ± 4.5 larvae,
35.2 ± 6.4 workers, and
4.2 ± 1.9 soldiers per colony
72 colonies with at least these numbers of individuals in their respective
caste were selected, with 12 replicates per treatment.
One
male
and
female
dealate

23. 23
Treatment Reproductives workers soldiers Eggs Larvae Loss(%) Gain (%)
Zero workers - - - - -
One worker + 3.9 ± 2.4 - + (Some missing) + (few) 78
2 workers + 4.12 ± 1.4 1 + + 73
5 workers + 9.7 ± 3.3 1-2 + (depleted) + (increased) 47
10 workers + 10.7 ± 3.8 1-2 + (increased) + 19
All workers + 9.2 ± 2.1 1-2 ++ (increased) ++ (increased) 301
The average number of workers gained was different among treatments (F = 15.6, P < 0.001)
Soldier ratio in the positive control treatment was significantly higher than in all other treatments
F = 7.08, P = 0.006
There was no difference in the total number of individuals between negative controls
and positive controls (t-test, P = 0.57).
Alloparental care is the cause for eusociality
Allo parental care is irreversible

24. 24
Pressure of nitrogen scarcity by Eusociality
Adaptations by termites for Nitrogen conservation
Thin cuticle
 Storage excretion of uric acid
 cannibalism
Nitrogen fixation mechanism
Eusociality
Increased
colony
growth
Require more
nitrogen for
initial growth
of the colony
Shift from
carbon rich
diet to
nitrogen rich
diet
Soil
 Soil is impoverished in organic compounds that are efficiently decomposed
 Enriched in recalcitrant materials, such as lignin, tannins, and other aromatic compounds,
that aggregate with carbohydrates and proteins to form humic and fulvic acids

25. 25
Cubitermes speciosus Sjiistedt,
Thoracotermes macrothorax (Sjostedt)
Crenetermes albotarsalis (Sjostedt)
Noditermes indoensis Sjijstedt
Collection of
species
Experimental
set up
Calibration Dissection Mounting Analysis
Mayombe rain forest, Republic of Congo
(Central Africa)
pH Profiles of the Extremely Alkaline Hindguts of Soil-Feeding Termites (Isoptera: Termitidae)
Determined with Microelectrodes
(Brune and Kohl, 1996)
Unidentified species of Cubitermes sp., was from a
location near Sarh, Republic of Chad (Central Africa)
Termitidae

26. 26
The hindgut of soil feeders has compartmentalized structure
The adaptation of soil feeding caused the elongation of gut increased gut pH

27. 27
Species Crop Foregut Midgut MS Hindgut Paunch Colon Rectum
Kalotermes flavicollos 5.2-5.4 6.8-7.5 5-7.5
Anacanthotermes ahngerianus 7.7 6-9 7.9
A. turkestanicas 7.7
Zootermopsis angusticollis 6.5-7 5.2-6.8 5.2 3-.8
Z. nevaddensis 5.5-6 7-7.5 7-7.5 7-7.5
Reticulitermes lucifugus 5.6 6.5-7.0 7.8 6-6.5 6.5-7.0
Reticulitermes sp.
Copotermes lacteus 3.8-4.4 6.8-7.4 6.8-7.4 6.8-7.4 2.8-3.2
Microcerotermes edentatus 8.8-9.6 8.8-9.6 >9.6 6-9.6 7.2-7.6 6.8-7.2 6.0-6.8
M. arboreus 6.5-7.5 7-8 7.5-8 6.4-7 6.5-7.0 6.5-7.0
Cubitermes severus 6-6.8 6.5-7.5 8-9.5 10-11 9-10 7.5-9.0 7-8
Procubitermes aburiensis 6-6.5 6.5-7.5 8-9 9.5-10.5 7.5-8.5 7.5-8.5 7-8
(Brien and Tor, 1982)

28. 28
Change in diet lead to loss of protozoa in higher termites (Termitidae)
Changes in gut physiology
Nitrogen rich diet starved the protozoans
•Fungal comb and Bacterial comb
Externalization of digestion (Macrotermitinae and
sphaerotermitinae)
Acquisition of new gut symbionts (Soil microbes)

29. 29
Evolution of Termite Symbiosis Informed by Transcriptome-Based Phylogenies
Collection of 55 termite species
Stored at -800 C
RNA and DNA isolation
Sequence alignment
MAFFT v7.305
Phylogenetic analyses
Method Parts/ sample used Sequence
RNA Phenolchloroform
procedure
Head of the 2-15
individuals
HiSeq 2500
platform
RNA RNeasy Plusmini kit Workers and soldiers HiSeq 2500
platform
RNA Phenolchloroform
procedure
Whole termite Nextseq500/550
DNA Dneasy blood tissue
extraction kit
5 workers HiSeq 4000
platform
Software: IQ tree 1.6.7
Method: Maximum likelihood
Bootstrap= 1000 replicates
(Bucek et al., 2019)

30. 30
Re-internalization of the wood feeding did
not invited the protozoa back to the wood
feeding Termitidae
Macrotermitinae and Spharotermitinae
evolved fungiculture and never lost
>75% Higher termites soil feeders
Molecular clock

31. 31
Diversity structure of the microbial communities in the guts of four neotropical termite species
(Vikram et al., 2021)
Northeastern Argentina
Cornitermes cumulans Microcerotermes strunckii Nasutitermes corniger
Termes riograndensis
mounds located in grasslands
Myracrodruon balansae
(hardwood)
Peltophorum dubium
(softwood)
DNA extraction Dissection of gut
16S rRNA (Bacteria and
Archaea)
ITS (Internal Transcribed
spacer)
rDNA (Fungal)
Bioinformatic tool
Qiime2 v2018.6
From live trees

32. 32
Bacteria)
(23 Phyla)
Cornitermes
cumulans
Termes
riograndensis
Microcerotermes strunckii
(hardwood)
Nasutitermes corniger
(softwood)
Spirochaetes 44% 20% 51 to 61 %
Fibrobacteria <13% 13%
Bacteriodes 13% 8%
Firmicutes 23% 32% 6% 8%
Bacteroides 13% 13% 8%
Archaea (<2%) (2 Phyla)
1. Euryarchaea
2. Bathyarchaeota

33. 33
Higher
termites
Completely lost
protozoa
Re-internalization of wood
feeding didn’t invited
protozoa in gut
Termitidae has acquired new gut
microbes compare to lower termites

34. 34
Cryptocercidae
Hodotermitidae
+Archotermopsidae
Stolotermitidae
Mastotermitidae
Kalotermitidae
Stylotermitidae
Serritermitidae
Rhinottermitinae
Psammotermitinae
Heterotermes
Copotermes
Reticulitermes
Macrotermitinae
Sphaerotermitinae
Foraminitermitinae
All other Termitidae
Termitidae
Rhinottermitidae
Lower
termites
Higher
termites
Acquisition
of
gut
protists
Proctodeal
tropholaxis
Allo
parental
care
Eusociality
Soil feeding
Externalization digestion
Loss of protozoa (54 MYA)
Wood roach ancestor
Summary
Complex faecal nest
structure

35. 35
Conclusion
Organism is result of what it
feed and where it live

36. 36
Thank You
For the Patience