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Water Bears of Baguio City: An Assessment of Limno-terrestrial

Tardigrade Diversity in GSIS Tree Park through Morphological


and Molecular Species Characterization
Cabanlong, J., Moldero, F., Nolasco, J.A.
Department of Biology, College of Science, University of the Philippines Baguio

INTRODUCTION

Tardigrades, commonly known as water bears, are classified as invertebrates and


micrometazoans composed of a body that is bilateral in symmetry, and lobopodous legs in four
pairs (Guidetti et al., 2017) (pinalitan ko na pero keep lang yung dialog box na may comments ni Commented [mm1]: I think this paper came from a
predatory journal. I personally don’t advice using these
sir, in case). These invertebrates are found in marine and freshwater ecosystems, as well as in kind of paper as references. There other articles from
semi-aquatic or limno-terrestrial environments (Middleton, n.d.). Limno-terrestrial tardigrades known tardigradologists which you can cite for these
statements. Refer to this (http://beallslist.weebly.com/)
live in lichens, mosses, liverworts, leaf litter, and soil and turf (Guidetti et al., 2017), specifically
in the collected interstitial moisture or trapped droplets of water (Miller, 1999) in leaves of Commented [mm2]: Same comment

mosses and thalli of lichens inhabiting trunks and branches of trees around the world (Lowman
et al., 2012). Aside from tree trunks and branches, tardigrade habitats can also be found in
epiphytes and in upper reaches of most tree species (Lowman et al., 2012).

Tardigrades can live in extreme and unpredictable conditions of living due to their ability
to undergo several dormancy strategies, one of which is cryptobiosis (Guidetti et al., 2017).
When tardigrades undergo cryptobiosis, their metabolism is suspended and they decrease in body
size up to about one-third of the normal, becoming what is referred to as tuns (Miller, 1999).
Tuns are capable of surviving periods of desiccation and freezing at any life-cycle stage (Guidetti
et al., 2017), and can withstand a temperature of 120°C, 1000 atmospheres pressure, and high
vacuums, as well as having resistance to compounds such as carbon dioxide and hydrogen
sulfide, and to X-rays and ultraviolet light (Miller, 1999).

According to Bartels et al. (2016), the checklist for the known tardigrade species showed
a total of 1,190 tardigrade species and subspecies as of 2013. Out of this number, 1,080 are
limno-terrestrial and 182 are marine. In their study, Bartels et al. (2016) estimated that the global
total of tardigrade diversity were 1,145 for limno-terrestrial and 936 for marine, indicating 87%
and 19% completeness of discovery, respectively. They concluded that tardigrades are both
poorly studied and relatively poor in species count.

In the Philippines, not much has been studied about the biodiversity of tardigrades,
especially in the Cordillera Administrative Region. Chang and Rho (1997) identified two species
of marine tardigrades in Palawan: Batillipes philippinensis and Florarctus kwoni and another two
in Mindanao: the Anisonyches deliquus and Anisonyches diakidius in 1998. Calud (2015)
sampled and identified tardigrades of genus Macrobiotus from lichen samples in University of
Southeastern Philippines. In 2016, Mapalo and co-workers completely described the first species
of limno-terrestrial tardigrade in the Philippines, which was named Mesobiotus philippinicus.
Recently this year, they again described a new species of limno-terrestrial tardigrade, from the
University of the Philippines Diliman in Quezon City. This species named as Mesobiotus insanis
is genetically close to Mesobiotus philippinicus.

Regardless of the overall abundance and diversity of the tardigrades, they have been
poorly understood. This is due to the fact that they are difficult to collect and culture. Also, they
apparently lack economic importance to human beings (Nelson, 2011). Given the status of
tardigrade fauna, the study on taxonomy and distribution of limno-terrestrial tardigrades in the
Baguio City Government Service Insurance System (GSIS) Tree Park pine (Pinus kesiya) trees
can be of remarkable contribution in identification, characterization, and classification of
tardigrades in the country. Species diversity is vital in the gene pool of a certain species. It
indicates whether an environmental stress could wipe out an entire population or an entire
population could withstand different disturbances. This means that the higher the species
diversity, the higher the chances of survival of a population. Since variation in morphology up to
the molecular level verifies the capability of the population to thrive, determination of this
characteristic of a certain population can determine the survival rate of organisms and can show
how this survival can affect the organisms. Understanding how tardigrades persist in extreme
environmental conditions can be a huge contribution to the scientific and medical fields as well
as in studies involving conditions or disturbances in highest degrees, which may lead to control
of species diversity if given enough attention (SquareSpace, 2013). Commented [mm3]: Personally, I made it a habit to
refrain from getting sources on a website. Maybe you
can look at this article?
With these facts in mind, this study aims to look into the existence and diversity of https://www.nature.com/articles/ncomms12808
limno-terrestrial tardigrades in Baguio City GSIS Tree Park near Baguio Convention Center,
specifically to:
1. characterize and identify the tardigrades based on morphological characteristics through
microscopy and imaging
2. identify and classify the tardigrades based on DNA sequencing.
3. determine if tardigrades can serve as environmental bio indicators

The study on taxonomy and distribution of tardigrades in the Baguio City GSIS Tree Park
pine trees can provide new information on the species diversity of Philippine limno-terrestrial
tardigrades. This new information could eventually lead to different discoveries such as
symbiotic relationships or species-specificity between tardigrades and different substrates of
mosses, lichens, and trees.

The focus of this study is only on limno-terrestrial tardigrades that will be found on the
associated lichens and mosses of pine trees in GSIS Tree Park near Baguio City Convention
Center. The study is to be conducted from October 2017 to May 2018. Assessment of the
distribution of tardigrades will involve the peripheral and inner areas of the tree park. Collected
tardigrades are to be characterized, identified, and classified only at morphological and
molecular levels using microscopy and imaging, and DNA sequencing, respectively.

REVIEW OF RELATED LITERATURE (char palang ‘to, copy paste lang mula sa intro,
pero yung limno-terrestrial studies lang esp yung kila Sir Mapalo yung ilalagay natin)

Calud (2015) sampled and identified tardigrades of genus Macrobiotus from lichen
samples in University of Southeastern Philippines. In 2016, Mapalo and co-workers completely
described the first species of limno-terrestrial tardigrade in the Philippines, which was named
Mesobiotus philippinicus. Recently this year, they again described a new species of limno-
terrestrial tardigrade, from the University of the Philippines Diliman in Quezon City. This
species named as Mesobiotus insanis is genetically close to Mesobiotus philippinicus.

METHODOLOGY

Sample Collection and Preparation


Commented [mm4]: We never tried sampling on pine
Samples of moss and lichens containing their vegetative structures will be collected from trees. Maybe you can try collecting from those trees.
I’m just curious whether there might be differences with
barks of pine trees in GSIS Tree Park near Baguio Convention Center. These samples will species composition if it’s from a gymnosperm or an
correspondingly be identified. The trees that will serve as substrates for collection will be chosen angiosperm. But then again, it can also depend on the
moss types and not the tree.
on two specific areas. These include the trees located on the periphery and trees located on the
Commented [mm5]: I’m currently using the methods
inner area of the tree park. The GPS coordinates of the areas of collection will be recorded. The stated in Stec et al’s paper
moss and lichen samples on the base of the trees will be scraped off using a pocketknife and (https://biotaxa.org/Zootaxa/article/view/zootaxa.4052.5
.1). You can download the paper here
placed in labeled containers or paper bags (Chang et al., 2015). (pacheck. Not sure if ganyan (https://www.researchgate.net/publication/285991655_
yung ibig niyong sabihin) An_integrative_description_of_Macrobiotus_paulinae_s
p_nov_Tardigrada_Eutardigrada_Macrobiotidae_Hufel
andi_group_from_Kenya).
In the laboratory, the collected samples will be soaked in Petri dishes with distilled water
It's what Daniel taught me when I was in Poland and
for about 12-24 hours (Chang et al., 2015 ; Miller et al., 2016). I’ve been using that method ever since. I think I’ve also
taught Lowelyn how to do this.

After submerging the habitat samples, distilled water and debris from each Petri dish will I recommend using glass equipment (pipettes, cylinder,
be drawn using a 1-mL pipette and transferred to a 25 x 50 mm labeled tray (Chang et al., 2015) petri dish) since tardirgrades tend to stick to plastic
more. It will be harder to collect them once this happen.
for examination under a 20x dissecting microscope (Miller et al., 2016).
Commented [mm6]: See comment above. I taught
Lowelyn how to collect tardigrades. Maybe you can ask
The tardigrades present in each tray will be extracted through the use of an Irwin loop her to teach you too.

(Chang et al., 2015) and an isogenic culture will be established following the methods by Mapalo Commented [mm7]: This culture method is best for
parthenogenic species. There are some species that
et al. (2016). First, a single female with mature oocytes will be isolated by transferring it in a might have eggs in them but still need to be fertilized
Petri dish lined with 2% agar made with KCM solution. The dish will be filled with a thin layer since they’re dioecious. I’m just saying this as a side
note that this culture method is not perfect for all
of distilled, deionized water (ddH2O). The culture will be checked daily for eggs and new species. However, you can always start with this. 😊 it
worked for me and Lowelyn’s cultures
individuals. A new female with mature oocytes from this culture will then be subcultured and
from then on will be fed with Chlorella sp. from Algae Research and Supply (Aquatic
Biotechnology Laboratory, National Institute of Molecular Biology and Biotechnology,
Philippines). From the subculture, individuals will be randomly extracted and separated into two
groups for the following analyses: (i) imaging and morphometry with phase contrast microscope
(PCM), and (ii) DNA sequencing.

Microscopy and Imaging

The following procedure will be done according to Mapalo et. al (2017). Specimens for
viewing under PCM will be mounted on microscope slides with a small drop of Hoyer’s medium
prepared based on Morek et al. (2016), and secured with coverslips. Slides will then be dried for
five days at 60°C in an incubator, after which they will be sealed with transparent nail polish.
Sealed slides will be examined under a Nikon Eclipse 50i phase contrast light microscope
associated with a Nikon Digital Sight DS-L2 digital camera.

Morphometrics and Morphological Identification

Tardigrades will be identified based on their visible characteristics using taxonomic keys
by Ramazzotti and Maucci (1983), Nelson and McInnes (2002), and Pilato and Binda (2010)
(Miller et al., 2016). The dichotomous key for the harmsworthi group by Kaczmarek et al.
(2011) will also be used for identification through description of tardigrade species (Mapalo et
al., 2017). Commented [mm8]: Tip: to distinguish tardigrades
from the order Parachela, I usually look at the claws
first. It will immediately tell you which superfamily they
Structures will be measured only if their orientation is suitable and the measurements will belong to.
be in micrometers (μm) (Mapalo et al., 2017). Sample size will be chosen following Stec and
colleagues (2016). The following dimensions will be measured as specified in the study of
Mapalo and co-workers (2017): body length, buccal tube length, buccal tube width, level of the
stylet support insertion point, macroplacoid length sequence, claw branch length, and point ratio.
Body length will be measured from the anterior extremity to the end of the body, except for the
hind legs. Length of the buccal tube and the level of the stylet support insertion point will be
measured according to Pilato (1981). For the buccal tube width, both the external and internal
diameters at the level of the stylet support insertion point will be measured. Macroplacoid length
sequence will be given according to Kaczmarek and colleagues (2014). As for the lengths of
claw branches, measurement will start from the the claw base (excluding the lunules) up to the
primary branch top including accessory points (Kaczmarek & Michalczyk, in press). The point
ratio which is the ratio of the length of a given structure to the length of the buccal tube, will be
expressed as percentage (Pilato, 1981). The “Parachela” ver 1.2 template from the Tardigrada
Register (www.tardigrada.net/register) will be used to handle the data on morphometry.
Gene Selection and DNA Extraction

The near complete 18S rRNA gene sequence (~1800 b) will be used for the molecular
analysis (Nichols, 2005). DNA extraction will be carried out according to the procedure by
Nichols (2005). Tardigrades from each isogenic culture will be isolated in individual glass slides
and air dried. The tardigrade cuticle will then be mechanically macerated using dental probes
cleaned beforehand with DNA-Away and rinsed in deionized water. A 10 μl solution containing
5mg/ml of Proteinase K will be added to the macerated cuticle and the sample will be treated to 3
freeze/thaw cycles at -20°C. The extraction mixture will then be transferred to a 200-μl
centrifuge tube and incubated at 55°C for one hour, 5 minutes at 95°C and then centrifuged at
2000 x g for 5 minutes. 10 μl of the supernatant will be used as template DNA. Commented [mm9]: If you can afford to buy a kit, I
would highly suggest using one since it’s less tedious. I
used the NucleoSpin kit based on this article
DNA samples of live and in anhydrobiotic stages of tardigrades will be extracted using (https://www.researchgate.net/publication/228448453_
Comparison_of_different_protocols_for_DNA_preparati
the NucleoSpin® Tissue DNA extraction kit. (or???) on_and_PCR_amplification_of_mitochondrial_genes_o
f_tardigrades)
Polymerase Chain Reaction

The procedure by Nichols (2005) will also be followed for this part of the methodology.
PCR amplification of template DNA will be carried out using primers specific for the 18S rRNA
gene (18S4 5'-GCTTGTCTCAAAGATTAAGCC-3' as the forward primer and 18S5 5'-
ACCATACTCCCCCCGGAACC-3' as the reverse primer). PCR reactions will be performed in
200 μl tubes using a thermocycler. The reaction cycle will be consisting an initial denaturing step
of 30 seconds at 94°C, followed by 35 cycles of 30 seconds denaturing at 94°C, 30 seconds
annealing at 45-55°C, and 60-120 seconds extension at 72°C. The PCR reactions will be
consisting of 1X final concentration of 10X PCR buffer, 2 mM final concentration of magnesium
chloride, 0.1 μM final concentration of each primer, 0.25 mM final concentration for each of
dATP, dCTP, dTTP, and dGTP, 10 μl of template DNA, and 1 unit of EnzyPlus 2000 Taq
polymerase in a final volume of 50 μl.

Sequencing

The DNA samples will be sent to Singapore (1st Base) for sequencing.

Comparison of Species Diversity of Tardigrades on Sample Areas

(pwede bang parang ito yung method for tardigrade as bio indicator chenes)

REFERENCES
Bartels, P. J., Apodaca, J. J., Mora, C., & Nelson, D. R. (2016 November 22). A global
biodiversity estimate of a poorly known taxon: phylum Tardigrada [Abstract]. Zoological
Journal of the Linnean Society, 178 (4), 730-736. https://doi.org/10.1111/zoj.12441

Calud, A. (2015). Tardigrades in Lichens and Mosses found in University of Southeastern


Philippines, Obrero. Retrieved from
http://www.academia.edu/13878945/Tardigrades_in_USEP_Obrero

Chang, C. Y. & Rho, H. S. (1997). Two new marine tardigrades from Palawan Island, the
Philippines. The Korean Journal of Biological Sciences, 1 (3), 419-423. Retrieved from
http://bionames.org/references/9d236216739ee67f31f9fa1aa504b391

Chang, C. Y. & Rho, H. S. (1998 June). Two Marine Tardigrade Species of Genus Anisonyches
(Heterotardigrada: Echiniscoididae) from Mindanao, the Philippines. The Korean
Journal of Systematic Zoology, 14 (2), 91-98. Retrieved from
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of_Genus_Anisonyches_Heterotardigrada_Echiniscoididae_from_Mindanao_the_Philipp
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Chang, L., Powell, D., Miller, W. R., & Lowman, M. (2015). Tardigrades of the canopy:
Evidence of stratification. Transactions of the Kansas Academy of Science, 118,
230-236. Retrieved from http://canopymeg.com/wp-content/uploads/2015/11/2015-
Chang_etal-Tardigrades-1.pdf

Devasurmutt, Y. R. & Arpitha, B. M. (2016 May). An Introduction to phylum Tardigrada –


Review. International Journal of Latest Technology in Engineering, Management &
Applied Science, 5 (5), 48-52. Retrieved from
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Guidetti, R., Kristensen, R. & McInnes, S. (2017). World Tardigrada Database. Retrieved
October 17, 2017 from http://www.marinespecies.org/tardigrada/

Guil, N. & Cabrero-Sañudo, F. J. (2007, September 9). Analysis of the species description
process for a little known invertebrate group: the limnoterrestrial tardigrades (Bilateria,
Tardigrada). In Vertebrate Conservation and Biodiversity (pp. 237-260). ISBN:
1402063202, 9781402063206

Lowman, M., Schowalter, T., Franklin, J. (2012, November 26). Methods in Forest Canopy
Research. ISBN: 0520953924, 9780520953925
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Mapalo, M. A., Stec, D., Mirano-Bascos, D., & Michalczyk, L. (2017 September 4). An
integrative description of a limnoterrestrial tardigrade from the Philippines, Mesobiotus
insanis, new species (Eutardigrada: Macrobiotidae: harmsworthi group). Raffles
Bulletin of Zoology, 65, 440-454. Retrieved from
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454.pdf

Middleton, R. (n.d.). Tardigrada (water bears, tardigrades). Retrieved October 17, 2017 from
http://www.biodiversityexplorer.org/metazoa/tardigrades/index.htm

Miller, W. R., Gallardo, L., & Clark, T. (2016 October 12). Do Water Bears Climb Trees Too?.
Treetops at Risk: Challenges of Global Canopy Ecology and Conservation, 307-311.
doi: 10.1007/978-1-4614-7161-5_30

Miller, W. R. (1999). Tardigrades: Bears of the Moss (About Tardigrades). Retrieved October
21, 2017 from http://www.pathfinderscience.net/tardigrades/cbackground.cfm

Miller, W. R. (1997). Tardigrades: Bears of the Moss (About Tardigrades). The Kansas School
Naturalist, 42, 3. ISSN: 0022-877X

Nelson, D. (2011). Current Status of the Tardigrade: Evolultion and Ecology. Retrieved on
October 30, 2017 from https://academic.oup.com/icb/article/42/3/652/724023/Current-
Status-of-the-Tardigrada-Evolution-and#12727086

Nichols, P. (2005). Tardigrade Evolution and Ecology. Retrieved on September 12, 2017 from
http://scholarcommons.usf.edu/cgi/viewcontent.cgi?article=1793&context=etd

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from http://www.fromthelabbench.com/from-the-lab-bench-science-blog/tardigrade-
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