Tardigrades in USEP Obrero

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Tardigrades in Lichens and Mosses found in

University of Southeastern Philippines, Obrero

Apple Rose B. Calud

University of Southeastern Philippines, 2015

Email address: [email protected]

Abstract

There are less than one thousand discovered,

identified, and classified tardigrades in the world. These

organisms belong to phylum Tardigrada, a phylum closely

related to nematodes and arthropods and live in terrestrial

and aquatic environments. Characteristics of tardigrades are

those shared from the two groups closely linked to them.

They exhibit unique characteristics such as cryptobiosis

that allows them to survive in adverse environments such as

extreme temperatures, pressure, and radiation. Although

their capability of entering into a cryptobiotic state is

interesting, tardigrades are poorly studied not only on the

local level but globally as well.

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Lichens and mosses, where tardigrades commonly thrive,

were collected in University of Southeastern Philippines for

the identification and determination of their abundance in

the two different substrata. Microscopy was conducted to

view tardigrades from the collected samples. Viewed

tardigrades were identified as member of the genus

Macrobiotus, under family Macrobiotidae, a eutardigrade. No

tardigrades were viewed from the moss samples. Thus, further

research through sampling of the two different substrata is

highly recommended.

Introduction

Phylum Tardigrada is one of the two phyla which have

less certainty of their phylogenetic position. Together

with Onychophora, tardigrades show characteristics which are

combination of arthropods and nonarthropods. Members of this

phylum are commonly called “water bears” and although

tardigrades show arthropod-like characteristics, they never

fitted in to be considered as members of phylum Arthropoda.

Tardigrades are relatively small with their size ranging

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from 50 μm to 1200 μm. Most of the species live in

freshwater and terrestrial environments specifically on

mosses and lichens although there are some marine species

identified. A tardigrade body is composed of a head and four

body segments which include ganglion in each segment and a

pair of limbs. Claws are found on each lobopodous leg of a

tardigrade. Circulatory and respiratory systems are lacking

in these organisms, however, digestive system is complete

and they have dorsal brain and ventral nervous system. Most

have separate sexes but hermaphroditic genera were also

recorded to exist (Shaw & Miller, 2013).

Water bears are referred as limno-terrestrial species

because they can survive in environments with varying

degrees of moisture. Tardigrade lifestyle differs; some

species live as commensal while others live as parasites of

other invertebrates (Pechenik, 2011). There are more than

900 species of tardigrades belonging to two major classes:

Heterotardigrada and Eutardigrada. Heterotardigrada has two

orders: Arthrotardigrada and Echinoiscoidea. Eutardigrada

has two orders: Parachela and Apochela. Echinoiscoidea is

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composed of terrestrial species characterized by members

with armoured cuticle while Arthrotardigrada is composed of

marine species with members having telescopic legs with

sucking disks and median cirrus on the head.

Arthrotardigrades can be sampled from intertidal zone to the

abyss. In these environments there is high species richness

with patchy distribution of small tardigrade population. An

interesting characteristic of tardigrades is observed during

low tide wherein they exhibit cryptobiosis or their ability

to reduce metabolic rate in order to withstand unfavorable

conditions or otherwise known as latency (Nelson, 2002). A

tardigrade on a latent state has reduced metabolism, growth,

reproduction, and senescence. One type of latency is

encystment that is observed in freshwater tardigrades which

can be sampled in lakes, streams, springs, ponds, and

temporary ponds. Terrestrial tardigrades are found in moist

habitats because one of the habitat requirements is an

alternate wet and dry periods and sufficient supply of

oxygen and food (Degma et al., 2014).

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Tardigrada is an ancient phylum as suggested by its

fossil records. To date, nearly a thousand species are

identified and there is more left undiscovered and

unidentified. In the study “The tardigrade Hypsibius dujardini,

a new model for studying the evolution of development” water

bears were studied in order to strengthen the proposition

that these organisms are potential models in the field of

evolution and development. Taxonomically, water bears are

members of the phylum of microscopic ecdysozoan animals, a

phylum closely related to the other development model

organisms Drosophila and Caenorhabditis elegans which makes

tardigrades as potential model organisms as well (Gabriel,

et al., 2007). Tardigrades were sampled for decades and

identified their genome and followed its cell divisions.

The general objective of the study was to determine the

presence of water bears in lichens and mosses. Specific

objectives were as follows:

1. identify tardigrades using taxonomic characters; and

2. determine the abundance of tardigrades in mosses and

lichens.

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Tardigrades have always been interesting organisms to

study as they are model organisms to understand the

arthropod phylogeny by determining their close association

with arthropods. This study would be an expansion of

knowledge considering that there are few studies on

tardigrades, their biology, ecology, systematics, and

identification of new species. Other than providing baseline

information on tardigrades, this study would be able to make

observations on the habitat requirements of tardigrades and

characterize further the biodiversity in lichens and

bryophytes.

Materials and Methods

Mosses and lichens were collected to view tardigrades.

Lichens were scraped from the tree barks while mosses were

collected from dirt, plant pots, trees, and rocks found

within the premises of the University of Southeastern

Philippines (USeP), Obrero campus. The university was

selected as the focus of the study because trees, where

lichens attached are found within the campus which made the

collection of samples easier. Moss collection was also

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easily conducted in the campus because they were present in

rock surfaces found just below the college building. As for

sampling lichens, they were scraped from the barks of a

7°5’9” N 125° 36’ 56” E

7°5’10” N 125° 37’ 2” E

7°5’ 9” N 125° 37’ 2” E7°5’ 11” N 125° 3’ 2” E

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mango tree situated near the clinic located 7°5’9” North

125° 36’ 56” East, additional lichen samples were collected

from 7°5’10” North 125° 37’ 2” East and from Acacia tree

located at 7°5’ 11” North 125° 3’ 2” East. The lichens

obtained were the crustose and foliose type, scraped from

the tree-to-eye level using spatula. Moss samples were from

rocks near the stairs of College of Arts and Sciences

building located 7°5’ 9” North 125° 37’ 2” East (Google

Earth, 2013). Forceps were used to pick mosses from the

surface of the stones.

Figure 1. Sampling stations in USeP. (Google Earth, 2013)

All collected samples were placed inside a paper bag

and were kept until dried. Lichen and moss samples were

placed in petri dishes and were hydrated with distilled

water to a depth of 0.5 to 1.5 cm. The petri dishes were

labeled with the date of collection, source of sample, and

specimen number. Petri dishes were stored under room

temperature for 48 hours prior to viewing. Individual

tardigrades were immediately documented under a compound

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microscope with 100x magnification and all observations were

recorded. Abundance which is defined as the number of

individuals was recorded every sampling (McGill et al.,

2007). Measurement was taken using the ocular micrometer

with the scanner objective with a calibration of 0.00211 μm.

Morphological structures such as the claws, mouthparts,

pharynx, and body were observed and recorded. Identification

was based on the identification key of Pilato and Binda,

2010. Photos of sample tardigrades were also sent to experts

for identification (Shaw and Miller, 2013).

Results and Discussion

Viewing of tardigrades was conducted in non-consecutive

sampling dates. Lichens and moss samples were viewed on

December 10, 13, 18, 2014 and January 6 and 10, 2015.

Several tardigrades were viewed under the microscope;

however, only few were properly documented. Between the two

sampling sources, documented tardigrades were from the

lichens collected.

Table 1. Individual tardigrade count in lichens and

mosses

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Source/Sample No. of

Individuals

Lichen 4

Moss 0

There were a total of 4 individual tardigrades

documented, however, there were no tardigrades viewed from

moss samples. This result was in contrast to a study

describing the suitability of bryophytes as tardigrade

habitat. Compared to lichens, limno-terrestrial tardigrades

most especially usually thrive in mosses because of the

capability of providing the appropriate environment for

their growth and survival. Mosses are capable of providing

three basic needs of tardigrades: adequate oxygen diffusion

provided by the structure of moss, the alternate periods of

drought and wetness, and the medium which provides

satisfactory food such as protozoa, nematodes, and the moss

itself (Glime, 2013). In addition, there were no evidence of

the habitat specificity of tardigrades and lichens are

considered as suitable habitats as well. Compared to mosses,

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preparation for viewing tardigrades was easier in lichens

than mosses because they were needed to be viewed

immediately unlike lichens which can be viewed even after 48

hours.

Consultation with expert Mike Shaw, a naturalist from

the United States of America and Paolo Fontoura, an

associate professor at the Department of Zoology and

Anthropology of the University of Porto in Portugal

supported the identification of the tardigrades as members

of the genus Macrobiotus. Macrobiotus belongs to class

Eutardigrada, under family Macrobiotidae together with 8

other genera (Miller, 1997). In the Philippines, meiofauna

in Bohol was studied and the study was able to determine the

abundance of tardigrades in the three sampling stations

established. However, tardigrades were only viewed in two

stations only and there was a total of 8 water bears

recorded (Hongayo et al., 2013).

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Figure 2. Macrobiotus labeled parts.

a.mouth;b.macroplacoids;c.pharynx;d.leg I;e.leg II;f. leg

III; g.leg IV; h.leg V; i.leg VI; j.leg VII; k.leg VIII

(Magnification:100x)

ab

cd

ef

g

h

i

jk

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This genus has 153 species of which 23 were newly

discovered. To mention a few: Macrobiotus anemone, M. pisacensis,

M. insuetus which were discovered in 2014, M. kristenseni in 2013,

and Macrobiotus sottilei in 2012 (Degma et al., 2014). In the

study on the tardigrades in Kansas, this genus has been

indicated as one of the genera a tardigradologists would

likely find in sampling tardigrades because they are

cosmopolitan (Miller, 1997).

They were described as tardigrades with smooth cuticle

and eye spots, buccal tube can either be wide or moderate in

width depending on what group they belong; hufelandi or

harmsworthi. Claws of this genus were described to be Y-

shaped and this group was considered as one of the most

difficult groups to be isolated (Miller, 1997). Figure 2

shows the different parts of Macrobiotus with their

corresponding labels. Posterior to the mouth of the

tardigrade is the buccal-pharyngeal apparatus which includes

the buccal tube, stylets, and pharynx. The buccal tube is

the slender tube which extends from the mouth and ends with

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macroplacoids. The macroplacoids were located inside the

pharynx, the oval-shaped structure (Pilato and Binda, 2010).

The body of the tardigrade is divided into five

segments: the head segment, three trunk segment, and the

caudal segment. The head segment include the mouth,

macroplacoids and pharynx labeled a, b, and c of Figure 2

respectively. Included in the trunk segment are the three

pairs of legs, each end up with claws; d, e, f ,g, h, and i

of the same figure. The caudal segment includes the fourth

pair of legs j and k of Figure 1. Moreover, Figure 3 shows

the Macrobiotus in dorsal, ventral, and lateral view (Degma

et al., 2014).

Identification of the specimen was based on the

morphological characters of the cuticle and other

structures. The cuticular appendages, cuticular plates, and

claws were used as keys. Macrobiotus is a eutardigrade and

members of this class are distinctively different from

heterotardigrades being considered “naked” because of their

lack of dorsal plates but some have spines instead (Degma,

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2010). Tardigrades, regardless of class have symmetrical

bodies (Figure 3a).

Figure 3. Different views of tardigrade. a.dorsal view;

b.ventral view; c. lateral view (Magnification:100x)

The figure above shows an active tardigrade, the body

is stretched and legs were extended. When inactive, it does

not show its legs and the body is shortened in a form of tun

(Miller, 1997). Ventrally, the buccal-pharyngeal apparatus

of the tardigrade is visible. Below are the actual photos of

a b c

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the buccal apparatus with the reference photo of the buccal

apparatus of M. hufelandi (Glime, 2013).

Figure 3. Buccal-pharyngeal apparatus. Fig.3c (Shaw and

Miller, 2013) (Magnification:100x)

Buccal apparatus is a good key in identifying the

members of Eutardigrada. As described by Degma in 2010,

buccal apparatus of Macrobiotus is usually supported by a

lamina located on the ventral side of the tardigrade. On the

a b c

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other hand, buccal apparatus are not commonly used in

identifying members of Heterotardigrada. As can be seen on

the diagrammatic illustration of the stylets of Macrobiotus,

the stylets are curved. Further description by Pilato and

Binda in 2010 is the presence of three macroplacoids and

another three microplacoids which are not distant from the

macroplacoids. Measurements of the specimens were recorded

and shown in Figure 4.

Specimen measured 0.60 μm in length and 0.02 μm in

width. Measurement was taken with the specimen under low

power objective (LPO). The body was transluscent, internal

organs are visible from the outside. Macrobiotus hufelandi, a

very common member of this genus grows at size enough to

allow them to walk in and through the bryophytes where they

usually live (Glime, 2013).

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Figure 4. Body measurements. a.body length;b.body width

(Magnification:100x)

Other species from this genus such as Macrobiotus crenulatus

measured 229.3–280.4 μm in length, this species was identified

in 2006 in Portugal. M. ramoli, was another species discovered in

Austria which measured 246-560 μm in length. (Dastych, 2005).

F

igure 5. Macrobiotus claw. (Magnification:100x)

a b

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In identifying the different families under class

Eutardigrada, claws are of primary importance. In

comparison, heterotardigrades differ from eutardigrades in

terms of the number of claws. The armored heterotardigrades

have maximum of four claws in each leg compared to

eutardigrades which only have 2. The primary and secondary

branches of the claw were the basis of referring double claw

to eutardigrades. Species level identification makes use of

the claws as well, with reference to the lunulae and

accessory points present at the tips of the branches of the

claw. Claws of Macrobiotus were unbranched, wherein the

primary and secondary branches are not divided forming Y-

shaped claws. Description by Pilato and Binda (2010)

identified the claws of Macrobiotus as hufelandi type. Basal

spurs in each basal section of the claw of this type are

absent. Each section is also subdivided into stems.

Conclusion and Recommendation

Tardigrades or the water bears are interesting

organisms to study because of their unique morphology they

share with two different groups of organisms: nematodes and

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arthropods. The capability of tardigrades to survive

unfavorable conditions by entering into quiescence is an

interesting topic for future studies which could be used in

the field of cryonics. Despite of their promising research

opportunities, only few species were identified and are

poorly studied in the Philippines. The tardigrades observed

could potentially be M. hufelandi because this species is the

most common tardigrade. Moreover, the habitats where

tardigrades thrive are poorly characterized in terms of

their suitability as habitat, most especially lichens where

samples obtained in this study were unanimously found. In

comparison to mosses, lichens were easily sampled.

Identification of tardigrades is based on the internal

organs such as bucco-pharyngeal apparatus which includes the

stylets, pharynx, and buccal tube. In order to identify

water bears in the genus level, claws are used as reference.

Microscopes are of primary importance when dealing with

tardigrades, thus, the use of phase-contrast microscope is

highly recommended for better identification.

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