Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No.

1) 49

non-GM soybean samples containing 5%, 2%, l %, 0.5%, 0.1 %, and less than
0.0.3% transgenic Roundup Reacly«l soybean. Detection limits for the 300-bp CamV
35Slm-epsps and the 173-bp nos tenninator gene fragments by the multiplex PCR
procedure used in this study were 0.5% and 1.0%, respectively. The three new
primers designed and tested in this study can now be used for multiplex PCR
detection of GM soybean containing the transgenes CamVIEPSPS and nos
tenninator and can be utilized in developing a commercial detection kit for such
transgenes in GM soybean.

Keywords: genetically modified (GM) soybean, Glycine max, multiplex PCR, PCR
detection limit

BIOLOGICAL SCIENCES

BSD No. I /
CHALLENGES IN THE STUDYAND CONSERVATION OF PHILIPPINE
RAFFLES/AS

*Julie F. Barcelona 1•4, MaryAnn 0. Cajano2, Leonardo L. Co3

and Danny S. Balete4

1
Philippine National Herbarium (PNH), Botany Division
National Museum, P. Burgos St., Manila, P.O. Box 2659;
2
College of Agriculture Herbarium, Museum of Natural History
University of the Philippines Los Banos, College, Laguna 4031
3
Conservation Intemational-Philippines,
6 Maalalahanin St., Teacher's Village; Diliman, Quezon City 1101;
4
Laksambuhay Conservation, Inc.
1024 l Mt Bulusan St. Umali Subd., Los Banos, Laguna;
Email: BarcelJF@hotmail.com

Rajflesia (Rafflesiaceae) is a genus of plants obligately parasitic to

Tetrastigma (Vitaceae ), lianas of the lowland rainforests in tropical and subtropical
Asia. To date, 21 species of Rajflesia have been described: one species in Thailand,
eight in Indonesia (five in Sumatra and three in Java), eight in Malaysia, and four
in the Philippines including the most recently described, R. mira, from Compostela
Valley in Mindanao. Our recent explorations in Luzon have added two more new
species. Three potentially new species, yet to be described, are currently known
from photographic evidence only. Once formally described, they would make the
Philippines the global center of Rafflesia with a total of at least nine species, the
50 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I)

highest species concentration relative to land area. The lowland rainforest, home
to rafflesias, is the most threatened due to deforestation and conversion to other
landuses. Because of their rarity, diversity, unique reproductive biology, and
ecology, studies on Philippine Rajflesia have not yet gone beyond discovery,
nomenclature, and mapping. Research on Rajflesia is currently being hampered
primarily by the generally tedious and slow permitting process of the Department
of Environment and Natural Resources or by the absence of competent botanists
at the time of discovery. In order to make sound conservation action plans for this
enigmatic genus and its host, collection and scientific studies of Philippine Rajflesia
must be supported by alt stakeholders. Furthermore, further explorations of the
lowland rainforests in the Philippines should be facilitated and collaboration
between Filipino and foreign parasitic plant experts should be encouraged. If the
scientific studies remain to be hampered by the lack of collecting permits and the
destruction and disappearance of the lowland rainforests in the Philippines is
unabated, perhaps the diversity of this plant group may disappear even before all
the species are discovered.

Keywords: conservation, lowland rainforests, parasite, Philippines, Rajjlesia,

Tetras tigm a

BSDNo.2
BUTIERFLY SPECIES IN LOWLAND AND MOUNTAINOUS LOCALITIES

Bonifacio F. Cayabyab

National Crop Protection Center, Crop Protection Cluster

College of Agriculture, University of the Philippines
Los Baftos, College, Laguna 4031, Philippines
Email: bfcayabyab@laguna.net

Butterflies have diminishing range due to habitat degradation brought about

by land developments and deforestations. Observations regarding butterflies in
lowland and mountainous locality will be elucidated in this paper. This study will
show the disparity of species in the said localities and the reasons for such contions.
A typical developed lowland like in Los Banos, Tarlac and Pampanga
neighborhood with few surviving flora will yield the following family and species:
Family Pieredae: Eurema hecabe, Catopsilia pomona, Catopsilia pyranthe,
Catopsilia scyl/a, Leptosia nina, Appias olferna, Family Lycaenidae: --Zizina otis
oriens, Eucrysops cnejus, Family Papilionidae: Papilio demoleus, Family
Nymphalidae: Junonia almana, Junonia hedonia ida, Hypolimnas bolina,
Melanitis leda leda, Family Satyridae: Bibasis gomata, Amathusia phidippus and
 Trans. Nat. Acad Sci. & Tech. (Philippines) Vol. 28 (No. I) 51

Erionota thrax. In contrast moWltainous surroWldings such as Mt. Arayat and

Mt. Makiling with remaining forest covers can yield more than one hundred species.
These species seldom venture outside the said mountains. The host plants land
use patterns and natural enemies affect the scarcity or abundance of a given
species.

Keywords: Butterflies, family, species, lowland, mountainous, localities

BSDNo.3
THEAMPHIBIANFAUNA OFMT.MALINDANG
MINDANAO, PHILIPPINES

l01ga M. Nuil.eza" and 2Fritzie B.Ates

I Department of Biological Sciences, College of Science and Mathematics

MSU-lligan Institute ofTechnology, Tibanga, lligan City
Email: olgamnuneza@yahoo.com
2Department ofBiology, University of the Philippines Mindanao, Davao City

The Philippines is one of the world's top 17 megadiverse countries, however,

it is not en exception to the general trend of forest destruction. Mt. Malindang is
one of the upland ranges where biodiversity has been severely threatened due to
forest loss. To assess amphibian fauna) species richness for better Wlderstanding
and management of critical resources, fieldwork was conducted in Mt. Mal indang,
Mindanao from October 2003 until December 2004 in different vegetation types
(mossy, montane, almaciga, submontane dipterocarp, lowland dipterocarp, mixed
dipterocarp and plantation forest as well as agroecosystem sites) from elevation of
120 meters to over 1700 meters above sea level. Primary data were collected
through opportunistic technique with the participation of Subanens (indigenous
people in the area) as local researchers. Assessment revealed 26 species of
amphibians, 11 of which are endemic species with seven found only in Mindanao.
Nine species are in the threatened category where eight are vulnerable and one
endangered. Philautus surrufus, a Mindanao endemic listed in the endangered
category was found to be abundant in the Mt. Malindang. All endemic species
captured were found to be forest dwellers. It is apparent from the results of this
present study that despite habitat degradation in Mt. Malindang, species richness
of amphibians is high where 26% of amphibians recorded in the Philippines and
74% recorded in Mindanao are found in Mt. Malindang, indicating the conservation
importance of this mountain range.

Keywords: amphibians, endemic, species, richness, Mt. Malindang

52 Trans ..Vat..4.md. Sci & Tech. (Phi/JpplHes) Va/_ 28 ('·lo_ I)

BSDNo.4
SURVEYOFENDEMIC,INDIGENOUSAND INIRODUCED (FOREIGN)
SPECIES INTHE FRESHWATER ECOSYSTEMS OFNUEVA ECLJA

Annie Melinda Psz-Alberlo•. Erlene S. Castro and

Vincent Lainard £. Flort$

Central Luzon State Universii:y, Science City of Munoz, Nueva Ecija

EmBil Address: ralbrtco@mozcom.com

The study was conducted to describe, identify, classify and to detennirn: the
occurrence and diversity of the endemic, indigenous and introduced (foreign)
species in the freshwater ecosystems ofNueva Ecija. It also aimed to detennine
the sources and level of impacts of environmental degradation in the freshwater
ecosystems ofNueva Ecija.
This survey was conducted in five freshwater ecosystems of Nueva Ecija,
namely, Talavera River in Talavera, Pampanga River in Palayan City, Tabuating
River in San Leonardo, Paitan Lake in Cuyapo and Pantabangan Dam in
Pantabangan. Three sampling stations were selected in each of the five study
areas and in each station ten quadrats measuring I0 m x J2 m were laid out randomly.
Freshwater fishes, mollusks and macroflora were collected in the five study sites.
An interview with the residents and a survey questionnaire was distributed ro the
30% of the total population of fishennen in each study area. An asseswe nt for
lhe environmental disturbance affecting the freshwater ecosystems ofNueva Ecija
was done by means of 11 checklist to survey the sources and !eve I of impact of
environment degradation of the freshwater ecosystens.
Twelve ( 12) species of indigenous and endemic species were identified and
classified in the different freshwater ecosystems ofN ueva Ecij a which include four
(4) species of fresh waler fishes, two (2) species of freshwater crus1accans, one (I)
species of freshwater mollusk and five (5) species of freshwater macroflora.
Leiopotherapon ph1.1nbeus (Ayungin or Lukaok) and GJoHogotiiiu giuris (Biya)
registered the highest percentage occum:nce among the identified freshwater
animal species. Ipomoea aquaJica got the highest percentage occurrence with
regards to macroflora. Pantabangan Dam registered the highest species diversity
value among the freshwater ecosystems surveyed.
Sixteen (16) introduced species are described, identified and classified in the
freshwater ecosystems ofNueva Ecija wh.ich include seven (7) species of freshwater
fishes, five (5) freshwater mollusks and four (4) freshwater macroflora. Nile tilapia
"Ti/apia nilotica" is the most common introduced freshwater fish while Pomacea
cunalic11lata also called golden snail is the most common introduced freshwater
mollusk in lhe selected freshwater ecosystems ofNueva Ecija. Moreover, Eichornia
crassipes also known as water lily is the most common introduced freshwater
 fran.r. Na1. !lead Sci. & Tech (Philippines) I iJI. 18 !So. I 1 53

macroflora. Paitan Lake got the highest species diversity of introduced species
which include fishes, mollusks and macroflora because the people near the lake are
dependent on those species as their source of food and source of income.
Based on the study, pollution such as animal wastes and solid wastes,
biological po!lution of introduced or alien species, recreational development,
establishment of the hydroelectric power plant, toxic chemical hazards and
recreational development have significantly contributed to environmental
degradation of the freshwater ecosystems in Nueva Ecij a.
At present, only a few number of indigenous and endemic species are present
and the introduced species domina1e the native species in the freshwater
ecosystems of Nueva Ecija. This condition may result into the extinction of the
endemic and indigenous species and to the destruction of ecological balance in
the freshwater ecosystems in the upcoming years as well as loss of biodiversity.

Keywords: Endemic species, indigenous species, introduced/foreign species, lake,

river, dam, biodiversity, environmental degradation, biological pollution

BSDNo.S /
ASSESSMENTOFMVCORRHIZALDIVERSrIY IN ABANDONED MINE
SITES IN TOLEDO, CEBU FOR BIOREMEDIATION

NeUy S. Aggangan 1, Nelson M. Pampolina2, Nina M. Cadiz' and

Asuncion K. Raymundtr

1
Nationa1 Institute of Molecular Biology and Biotechnology
1
Departmen1 of Forest Biological Sciences
College of Forestry and Natural Resources
1
Instirute of Biological Sciences College of Arts and Sciences
University Philippines Los Bru'!os, College, Laguna 4031
Email: nea@laguna.net

A survey of mycorrhizal diversity 1:1.!lsociated with selected plant species

within the 35,000-hectare Atlas Mines in Toledo, Cebu, was conducted with the
goal of rehabilitating abandoned copper mines through bioremediation by utilizing
indigenous plants and mycorrhizal fungi. The Atlas Mine site is generally covered
by vegetation classified as a disturbed grass-shrub-agroforest plant community.
Plants growing in marginal and stressed environments similar to lhat in mine sites
~ual\y are associated with mycorrhizal fimgi. P1811ts were identified and rhizosphere
soil (including fine roots) were collected in five sites delineated by the mining
company where ten samples were randomly collected from each site. Mycorrhiza I
colonization was 11Ssessed aft.er the roots were cleared wi1h potassium hydroxide
54 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I)

and stained with tryphan blue. Mycorrhizal spores were separated from the soil
using the wet sieving and decanting procedures and counted following the grid
line intersect method.
In the survey conducted, out of the 50 plants, only five (i.e. Lycopodium,
Saccharum spontaneum, Nephrolepis, Acacia mangium and Stachytarpheta
jamaicensis) collected from two sites showed colonization by vesicular-arbuscular
mycorrhizal (VAM) fungi, with infection ranging from l 0 - 100%. All roots of
kandi-kandilaan (Stachytarphetajamaicensis) were colonized solely by Glomus
sp. Kamachile (Pithecellobium dulce) harboured the highest spore density (2,575
spores/plant/30g soil), consisting of Glomus, Acaulospora and Entrophospora.
Datiles (Muntingia calabura), on the other hand, was the only plant associated
with Glomus, Gigaspora, Scutellospora, Acaulospora and Entrophospora, but
with low spore population. Glomus was the most prevalent among the YAM fungi.
Fruit bodies of ectomycorrhizal (ECM) fungi: Pisolithus, Scleroderma, Thelephora
and Bolettellus were found under Acacia auriculiformis, A. mangium, Eucalyptus
urophylla and E. camaldulensis. Pisolithus was the most prevalent. These VAM
and ECM fungi can be isolated and mass-produced as inoculants for the successful
rehabilitation of mine sites. Nursery and field trials have shown that mycorrhizal
fungi can enhance plant growth, promote survival in infertile and acidic soils and
tolerance to heavy metals.

Keywords: mycorrhizal diversity, YAM fungi, ECM fungi, rehabilitation, mine sites

BSDNo.6 /..r
GROWTHAND HEAVYMETAL~ORPTION OF MYCORRHIZALAND
NON-MYCORRHIZALJATROP/l4 CURCAS LIN MARGINAL
GRASSLAND AND MINE WASTE SOILS

Eusebeia Joy B. Mendoza', Clarice Meryl M. Ocampo 1 and

Nelly S. Aggangan 2

1
University of the Philippines Rural High School and
2
National Institute of Molecular Biology and Biotechnology (BIOTECH),
University of the Philippines Los Bafios College, Laguna 4031
Email: nea@laguna.net

This study determined the effect ofmycorrhizal inoculation on the growth of

Jatropha curcas L. in 9 mine waste soils and compared it with that in marginal
grassland and garden soil. Soil pH ranged from 2.4 to 6.8 and Cu concentration of
3.5 to 200 mg/kg soil. One-week old seedlings were inoculated with mycorrhizal
fungus Gigaspora margarita. The initial height of the plants was measured a week
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I) 55

after transplanting and the succeeding height measurements were done once a
week for ten weeks. At harvest, root collar diameter, leaf area, plant dry weight,
mycorrhizal infection and Cu and P concentration and uptake of different plant
parts were determined. Results showed that out of the 11 soils studied, only seven
sustained growth of Jatropha throughout the ten-week period. Plants grown in
garden soil exhibited the best growth and highest P uptake, followed by those
grown in a grassland soil. The poorest growth was observed in plants grown in
mine soils collected from Antamok, Benguet and Paracale, Camarines Norte.
Mycorrhizal inoculation promoted better growth of Jatropha in all soils except in
garden and grassland soils. Phosphorus and Cu concentrations and uptake were
highest in the leaves and lowest in the roots which were comparable with that in
the stem. This implies that mycorrhizal Jatropha grew better in heavy metal sites
and may contribute more in the extraction of Cu and other heavy metals in grassland
and mine waste soils than the non-mycorrhizal ones. Moreover, better growth of
mycorrhizal plants will definitely mean more fruits where biodiesel can be extracted.

Keywords: heavy metals, mycorrhiza, Jatropha curcas, mine waste soil, marginal
grassland, copper

BSD No. 7
MYCORRHIZALFUNGIAND HEAVY METAL TOLERANCE OF
FAST GROWING REFORESTATION TREE SPECIES

Nelly S. Aggangan 1 and Ma. Concepcion P. Segismundo2

'National Institute of Molecular Biology and Biotechnology (BIOTECH)

University of the Philippines Los Banos, College, Laguna 4031
2
Lipa City National Science High School, Lipa City, Batangas
Email: nea@laguna.net

Rehabilitation and reforestation of heavy metal contaminated sites can

significantly contribute to our country's economical, agricultural and ecological
stability.
Three fast growing tree species were used in the experiment namely: Acacia
mangium, Acacia aulacocarpa and Swietenia macrophylla and inoculated with
either Gigaspora margarita, Glomus etunicatum, a commercial mycorrhizal
inoculant "Mykovam" and mycorrhizal fungi from mine site coded as Paracale
isolates. Mykovam is a soil inoculant comprising of G margarita, G etunicatum
and G macrocarpum whereas Paracale isolates were a mixture of Glomus,
Entrophospora, Scutellospora and Acaulospora.
56 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. 1)

The study was conducted to determine heavy metal tolerant fast growing
reforestation tree species, and to determine the growth promoting and ameliorating
effects of mycorrhiza in fast growing trees grown in mine waste soil. The experiment
was one in a Randomized Complete Block Design with 10 replicates.
Inoculated A. mangium seedlings showed higher height and biomass than
the uninoculated ones. Mycorrhizal A. au/acocarpa, showed higher height,
biomass and phosphorus uptake than non-inoculated ones. Swietenia
macrophylla seedlings were significantly affected in their height and biomass but
the phosphorus content was higher than the non-mycorrhizal counterpart.
Inoculation with mycorrhizal fungi enhanced the heavy metal tolerance of A.
mangium, A. aulacocarpa and S. macrophylla and could be potential species for
the rehabilitation and reforestation of heavy metal contaminated areas.

Keywords: Acacia mangium, Acacia aulacocarpa, mycorrhiza, copper, heavy

metals, mine sites, phosphorus, Swietenia macrophylla.

BSD No. 8
ANALYSISOFHEAVYMETALUPTAKEOFSOMEPOTENTIALPLANTS
FORPHYTOREMEDIATIONINANABANDONED MINE AREA

Nina M. Cadiz 1, Nelly S. Aggangan 2 Nelson M. Pampolina 3 and

Asuncion K. Raymundo 1

1
Institute of Biological Sciences, College of Arts and Sciences,
2
National Institute of Molecular Biology and
3
Department of Biological Sciences
College of Forestry and Natural Resources
University of the Philippines Los Bafios, College, Laguna 4031
Email: nm_cadiz@yahoo.com

Phytoremediation, is offered as a "green" solution to environmental problems

and may be applied in situ or ex situ, to soils, sludges, sediments, other solids, or
groundwater. The study primarily aimed to investigate the tolerance mechanism of
selected dominant plants as·potential phytoremediatiors thriving in an abandoned
mine area in Toledo, Cebu.
Tissue analysis of the various plant organs of silver fem, malatungaw and
Japanese acacia revealed their various capacities to take up high concentrations
of copper (Cu), lead (Pb), cadmium (Cd) and zinc (Zn), the usual HM contaminants
in a copper and gold mining sites. Japanese acacia and silver fem showed high
concentrations of Cu in the roots. Japanese acacia, as a nitrogen fixer, has rhizobial
symbionts while the silver fem has mycorrhizal root association. In spite of high
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. 1) 57

concentration (43 5.17 mg kg- 1) of Cu in the roots of Japanese acacia, it was notable
that the metal was not trans located in the fruits. Moreover, the levels of Cd, Pb
and Zn in the fruits were very much below the allowable limits. Malatungaw, on
the other hand, showed the lowest uptake of the HMs among the dominant
species in the area, and thus, may be considered as a true excluder of Cu, Cd, Pb
and Zn.

Key words: heavy metals, tolerance, uptake, phytoremediation, abandoned mine

area

BSDNo.9
PHYTO,MONITORING OF Rhoeodiscolor AS DETERMINANT OF AIR
QUALITY IN TWO DIFFERENT SITES

Erwin P. Elazegui , Vanessa R. Garcia , Timothy Z. Riva

College of Science, Technological University of the Philippines, Manila

Email: ngsepe@yahoo.com

Phytomonitoring is the use of plants as determinants of environmental

pollution. Plants respond to pollutants in the form of changes in certain parameters
such as chlorophyll level , leaf-extract pH , relative water content and stomata!
density. These changes can be measured and used as a reliable indicator of exposure
to airborne pollutants . Phytomonitoring as its is called can thus be used as a
possible alternative and substitute to the usual physico-chemical methods and
costly instruments used in air quality monitoring.
The study focuses on the potentials of Rhoeo discolor as biological indicator
of air quality by phytomonitoring methods. Sample leaves were collected in two
sites , Laguna and Manila . Four phytomonitoring methods were used : total
chlorophyll content, leaf extract pH, relative water content and stomata! density.
Results showed that sample leaves from manila have lower values of total
chlorophyll content , 5.11 mg/g ; leaf extract pH 5.55 ; relative water content ,
93.25%; and stomata! density, 19.82 stomata per cm2 compared to those from
Laguna total chlorophyll content I 0.0 mg/g, leaf extract pH 6.68; relative water
content 99.08% and stomata! density 29,519 stomata per cm2 . Based on the
results, Rhoeo discolor is a good biological indicator of air quality.

Keywords: Phytomonitoring , Rhoeo discolor , biological indicator , stomata!

density
58 Trans. Nat. Acad. Sci, & Tech {Phihppines) J."o/_ 28 (No I)

BSD No. to
DEVEWPMENTOFASTANDARDTOXICITYTESTPROTOCOLFOR 11IE
MARINE ENVIRONMENTUSINGTIIE BLACKTIGERSHRIMP,PENAEUS
MONO/JON

Emilyn Q. Espiritu*, Katrina Lynn 8. de Guzman

and Carlos Celso V. Quevedo

Department of Envirorunenta! Science

Ateneo de Manila University, Loyola Heights, Quezon City 110 8
Email: eespiritu@ateneo.edu, katjadeguzman@yahoo.com.ph;
morisato54@yahoo.com
Biological assays are an essential tool for the determination of potential
adverse effects ofxenobiotics in the environment. However. many of the protocols
for toxicity testing use temperate organisms and could not be directly applied to
Philippine studies. The study explored the potential of the marine Black Tiger
shrimp, Penaew monodon, as a test organism for toxicity testing. 'The investigations
focused on the following aspects: (a) standardization of\aboratol)' conditions; {b)
detennination of the appropriate larval stage for use in toxicity testing; tc) selection
of the appropriate reference toxicant; (d) comparison of test sensitivity between
the Penaeus monodon test and the Standard Artemia ji·ancisr::ana toxicity test;
and {e) application of the developed test to environmental monitoring.
The results showed that the post-larval stage of the shrimp is the most
suitable stage to be used for toxicity testing in comparison to the protozoeal and
mysis stages. Moreover, among the toxicants tested (i.e. potassium dichromate,
copper sulfate, and mercuric chloride). the organism exhibited the most stable
response to potassium dichromate, KFrp 7, which was consequently chosen as
the reference toxicant. lnter-ca!ibnnion tests done with the standard Artemia toxicity
test showed that the Penaeus test is comparable in sensitivi~ with the ArTox test.
With these promislng results, the Penaeus monodon test can be a useful addition
in the Iist of wxicity tests for the marine environment which are app 1icab le to the
Philippine conditions.

Keywords: bioassays; xenobiotics; toxicity testing.; Penaeu.~ monndon: Artenzia

franciscana: potassium dichromate, post larva.
 Trans. Nat Acad_ Sn. & Tech (Philippirws) Vol 28 (Na fl 59

BSD No.11
GROUNDWATER QUALITY ASSESSMENT
IN PAYATAS DUMPSITE, PHILIPPINES

Glenn L. Sia Su

Biology Department, College of Science, De La Salle University- Manila

Email: glss76@lycos.com

Groundwater is an important resource providing man with numerous uses.

However, it has often been neglected and overly exploited. The concern over
today's contamination of groundwater and surface water sources resulting from
open dumpsites had been identified as a potential threat to Metro Manila's water
supply. The study assessed and compared the groLindwater quality of fourteer.
( 14) selected we l\s continuously used in the "with." (Payatas) and "without
dumpsite" (Holy Spirit) areas of Payatas estate, Philippines. Water quality
monitoring and analyses of the bio-physico-chemical parameters: pH, total
suspended solids (TSS), tot.al dissolved solids (TDS), total coliform, turbidity,
conductivity, salinity, nitrate-nitrogen, sulfate, color, total chromium, total lead
and total cadmium were carried out for six consecutive months from April to
September 2003, covering dry and wet seasons. Results showed that most of the
groundwater quality parameters in both study areas were within normal water
quality standards of the DENR-EMB and the DOH except for the observed high
TDS (> 1000 mgll), TSS (>50 mg/L), total coliform (MPN > 0) and low pH levels ( <
6.5). No significant differences in the following parameters: nitrate-nitrogen, total
cadmium, total lead, total chromium and total coliform were observed between the
"with" and "without dumpsite" areas. The parameters: TDS, turbidity, conductivity,
salinity and sulfate concentrations in the "with dumpsile" groundwater sources
were significantly higher. The signi !icam differences at the levels o fthe pollutants
strongly indicate the existence of pollution at the Payatas open dumpsite.
Continuous water quality monitoring. is encouraged. lncreasing the frequency of
sampling and analyses on the study areas are needed to effectively monitor impact
of dumpsites on the environment and human health.

Keywords: water quality, groundwater, dumpsite, Payatas, Philippines

60 [runs. Not. Ayid Sci. & Tech. (Philippines; Jo/_ 28 (!>'o. {)

BSDNo.12
ECONOMIC AND BIOJEOINOLOGICALPOTENTIALOFMUNICIPAL
SOLID WASTE: LESSONS FROMDUMPSITE SELECTION PRESSURES
AND EMERGENCE OF UNIQUE MICROBIALSfRAINS

Franco G Teves*, Suzanne E. Go and Aniline D. Sacdalan

Molecular Genetics end Microbial Technology Laboratory,

Department of Biological Sciences, College of Science and Math em a tics
MSU-lligan Institute ofTechnology, 9200 Uigan City
"'Emai I: franco _teves@yahoo.com

We focused our study on a ten-hectar.:, thirty four-year old dumpsite in

11 igan City, where approximately 120 tons of non-segregated municipal solid waste
are dumped daily. A consequence of the diverse compounds present is the selection
ofa unique microbial biodiversity.
Four composite sampling sites were identified representing 1.0% of the whole
dumpsite area. Soil samples were obtained and processed for the isolation of
facultative and aerobic bacteria and fungi. Isolates were pre-screened for their
production of bioacti ve compounds by testing lheir metabolites for anti-bacteria!
and anti-fungal properties. Selected microorganisms were tested further for lhe
production of DNA-binding metabolites using a two-dimensional thin layer
chromatography (2D-TLC) set up.
Of the 26 bacterial isolates, four exhibit potential for an industrial application,
with nine of the 73 fungal isolates also showing the same. While common microbial
srrains can be purchased from culture collections at a very low price (PhP 250.00 to
PhP 300.00), special and industrial strains can run from PhP I 0,000.00 to PhP
20,000.00 per strain, based on average international prices. Granting that the ra1io
we obtained from our two-step screening is the actual picture when applied to the
whole dumpsite area, we calculated a total of roughly J ,300 microbial isolates with
diverse capabilities waiting to be discovered in the ten-hectare site. Jfwe place tbe
number of industrially useful srrains to be only l 0% of the total, we would get 130
special ~mains. Translating these figures to commercial value of lhe microbial
strains would give an estimate of PhP 1,300,000.00 to PhP 2,600,000.00 from sales of
the cultures alone. This new paradigm should be considered in order to give added
value to what we refer to as municipal solid waste.

Keywords: municipal solid waste, microbial biodiversity, DNA-binding, 2D-TLC

 Trari:i'. Na/, Acad. Sr:i. & Ter:h f PhibpµinesJ lo/_ 28 (No. I1 61

BSD No.13/
LTTILIMTION OF AN INDIGENOUS DYESTUFF FROM Barella rubra
(ALUGBA.TI)AS MJCROBIOLOGICA.LSfAJN

Lorna T. Enerva, Winrich A.. Bati, Arby B. de Jesus, Angeli to T. Uldo 1

and Julius L. Leallo Jr. 1

College of Science and SciTech Rand D Center'

Polytechnic Unh•ersity of the Philippines
Philippine Textile Research lnstirute 1
Email: lomaenerva@yahoo.com

This study was undenaken to extract anthocyanin from Base!la rubra berries
and utilize the extract as microbiological stain. lt is- an inexpensive, indigencous
and abundant raw material.
The alugbati berries were macerated in a blender and extracted with 1% HCl
in 95% methanol. The extract obtained was filtered and then concentrated. Thin
!ayer and column chromatography methods were used to isolate and purify the
anthocyamin. The samples were analyzed using infrared spectra and ultraviolet
spectra. FT·IR re.,,ea\ed the presence of a hydroxy I group which is prominent in the
structure of anthocyan in pigment at 3385cm· 1• The C=-0 stretching for aromatic
ring were indicated by a peak at 1635.20 cm·', 1513.38 cm-i for C"'O bending,
1439.91 cm· 1 for 0-H bending, 1207.42 cm·1 for C-0 srretching, J l 51.92cm· 1 for
alkane and I I00. 77 cm- 1 for C-C stretching. The structure of anthocycanin was
further established by the t! maximum of the ultra.,,iolet spectrum at 510, 0 nm.
For the application, the crude extract was used as a stain for Staphyloccus
aureus, a gram positive bacteria and Escherichia coli, a gram negati...e bacteria.
The staining process for the microorganism used mordants like potassium alum,
cak ium oxide and copper sulfate for fixing the color. Only copper sulfate and lime
responded positively as a mordant that gave favorable outcome in fixing the color
of alugbati, The samples were screened based on tht criteria of color retention and
evenness. The structure of the microorganisms with respect to shape and size and
certain cellular components were identified using a microscope and
photomicrographs. The alugbati extract produced stain that was comparable with
synthetic stains like crystal violet and safranin and can. therefore, be used as an
altemati"e stain.

Keywords: anthocyanin. microbiological stain, mordant, dye.

62 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I)

BSDNo.14
PLANT DIVERSITY AND ABOVE-GROUND CARBON BUDGET OF
BROADLEAFSPECIESALONG NORTHEASTERN SLOPE OFMT.
MAKILINGFOREST, LUWNPIIlLIPPINES

1
DIXONT. GEVANAand 2 NELSONM. PAMPOLINA

1
Environmental Forestry Program. College ofForestry and Natural Resources
Email: enfor@laguna.net,
2
Department of Forest Biological Sciences,
College ofForestry and Natural Resources, University of the Philippines
Los Banos, College, Laguna 4031;
Email: nelmpampolina@yahoo.com

This study aims to assess the plant diversity resources and carbon budget
along elevation gradient in Mt. Makiling forest. Three elevation ranges were
identified along the northeastern slope of the mountain: low elevation (50-300
mas!), middle elevation (301-700 mas!), and high elevation (701-1114 mas!). Within
these ranges, vegetation samplings for broadleafwere done on tree, intermediate,
and wildling layers using the Quadrat Sampling Technique. A total of 110 species,
75 genera and 35 families were identified. Family Moraceae and genus Ficus have
the highest number of occurrence in all elevation ranges, while Swietenia
macrophylla was the most abundant species that had occurred from low to middle
elevation. Vegetation and biometrics were generally denser in low and middle
elevation while partially open and low on high elevation. The ecological values
also showed that density, frequency and dominance were higher on these elevations
where Parashorea malaanonan and Pterocymbium tinctorium had the highest
Importance Value (IV) recorded. The diversity values in all ranges were generally
low to moderate. The highest diversity was observed in high elevation that exhibited
a trend of increasing from low to middle elevation then decreasing from middle to
high elevation. Similarly, aboveground biomass and carbon density exhibited a
same trend and found highest at the middle elevations where plantations of S.
macrophylla and Dipterocarps are located. The highest estimate for carbon density
was 451.62 ± 50.07 at 400 mas! while lowest at the 900 mas! with 94.58 ± 24.12.
Overall, plant diversity resources and the corresponding potential carbon
sequestration are important ecological values that should be considered as input
in sustainable management of Mt Makiling forest.

Keywords: Plant diversity, broadleaf species, carbon budget, Mt Makiling

Forest
 Trans. _"iat. Acad Sci. & Trch !Phll1ppines! J'o/_ 28 (,Vo. I 1 63

BSD No. 15
DEVELOPMENT ANDQUALITYCONTROLOF A 10% TINCTURE OF
l,POMOEAMURICATA L. (JACQ.) (CONVOLVULACEAE)

*Sandra Cases Sy1.u and Marina 0. Osi1

1
Facuhy of Pharmacy, iGraduate School,
1
Research Center for the Natural Sciences,
University of Santo Tomas, Espafl.a, Manila
Email: scsy@mnl.ust.edu.ph

Phytochemical and clinical studies have shown that the seed of Jpomoea
muricata L. possessed promising antibacterial activities and analgesic effects.
This inve-stigation dealt with the quality control of a tincture developed !Tom the
crude ethanolic extractofthe dried seeds of /pomoea m1Jrica111 L. for purposes of
standardization in the interest of monograph preparation and drug formulation.
The 10% tincture is a thin, yet low liquid turning brown to dark brown with age;
with a tamarind-like and slight alcoholic odor. lt has a specific graviry of about
0,9185 and a pH of 5.70. It had a mean potencyof9.89%(specification: 9-l 1%w1
v) when assayed by a spectrophotometric method developed in the course of the
research. Such method was validated for precision, accuracy and linearity.
The assay procedure may not be stability-indicating since regression analysis of
accelerated stability data revealed inconsistencies of corre-lation data that made
them unsuitable for predicting shelflife using Arrhenius equation. ln view of this,
a long-term stability study remains the best approach to determine stability. Based
on real-time data of 12 weeks, the tincrure has a predictive she] flife of ! 8.5 weeks,
As final output of this study, a monograph on the finished product "Tinctura
lpomoea Muricatae Semen" (Tonkin Seed Tincture) has been generated.

Keywords: /pomoea muricara L., tincture. TLC profile, ultraviolet spectra, validation,
stability, monograph
64 Trans. Nat. 1/ cad Sci. & Tech !Plulippi11esJ Vol 28 !No I J

BSD No. 16
SUPERCRITICAL(SC}CARBON DIOXIDE (C01) EXTRACTION AND
FRACITONATION OFBJOACTIVE METABOLITES
FROM Ganoderma applonatum

James G. Mercado 1, Aldrin Wendell H. Suarez1, Roberto M. Malaluan1

and Franco G. Teves 1'"

t Molecular Genetics and Microbial Technology Laboratory,

Department ofBiological Sciences College of Science and Mathematics

1
Department of Chemical Engineering Technology,
School of Engineering Technology
MSU-lligan Institute ofTechnology, 9200 lligan City;
*Email: ITanco _teves@yahoo.com

Traditional solvent extraction of bioactivc compounds from biological

materials such as the shelf fungus Ganoderma applanat1.1.m, usually requires an
1111ay of extraction solvents. The use of supercritical-carbon dioxide (SC-CO,)
demonstrates a time-and-cost efficient procedure for extraction of polar and non-
polar components of G applanatli.m.
Fruiting bodies of the shelf fungus were collected from a semi-forest area in
Tandag, Surigao del Sur, cleaned of extraneous materials, and ground using a pre-
washed laborntory mil I. Ex.traction of the polar fraction was done by setting the
temperature at 40 ~c. a pressure of300 atmospheres latrn) and a fiow rate of 0.5 mil
h at norm a1conditions lT ~ 25°C, P - l ann ). The non-polar fraction was obtained
using a temperature of 40 °C, a pressure of 120 atm, and a tlow rate of0.5 milh. Total
extraction time was four hours each, for the polar and non-polar ITactions.
More than 50 ml each of the polar and non-polar ITactions were obtained
from separate 100 grams ground samples ofG applanatum, sufficient volume for
use in the various bioassays. Overall. the extraction period using SC-CO, was
much shorter (total of eight hours) compared to the traditional solvent extraction
(36 to 48 h). Moreover, usually only amounts in milligrams are obtained after
concentration by rotary evaporation. With SC-CO,. concentrated amounts in
solution are readily obtained. Additionally, since toxic and obnoxious chemical
solvents are no longer employed, SC-COt proves to be a cheaper and safer way to
fractionate metabolites and ex.tracts for bioactivity testing. In so far as literature is
concerned, this is the first report on the use of SC -CO: extraction of metabolites
from G apphmatum.

Keywords: Supercritical-CO,, Ganoderma applarwfllm, bioactive, bioassay,

metabolites ·
 Trans. ,\'al Acad. Sci. & Tech. (Philippiries/ Vol 28 rNo Ii 6S

BSDNo.17
EVALUATION OF LEAF EPIDERMAL CHARACTERISTICS OF SOME
PLUMER/A SPECIES

Vivian S. Tolentino, Haziel Adrielle P. dela Roso*, and Vivian A. Panes~

Department of Biology, Schoo 1of Science and Engineering

Ateneo de Manila University, Loyola Schools, Quezon City
Email: vtolentino@ateneo.edu

Plumeria species commonly called Kalachuchi are generally planted for

ornamental purposes, and are now widely cultivated throughout the country
because of their resilience, robustness and versatility in different types of
environments. In some coumries, it is cultivated for its fragrance for perfumes.
but is rarely cultivated for its medicinal properties. Plumerias are commonly found
in sacred places. such as churches, monasteries, temples and even in schools_
Nowadays, they are usually found in roadsides and in parking areas, It is for this
reason that the main objective of this study is to look into the leaf epidermal
characteristics of the plant which may be responsible for the wide adaptability of
this plant in different environments. The main objective of the study is to characterize
the leaf anatomy oft he epidermises of the different varieties of P\umeria variants.
The different cells of the epidermises, such as the hairs or trichomes, stomata!
apparatus, its location, stomatal index and type were studied. Histological
techniques such as leaf dearing, epidermal imprints and cross-sections of leaves
using fr.ee hand, sliding and rotary microtome were used. Ulustrative slides were
used for anatomical characterization of different epidermal structures, observed
and photomicrographed using the BH-epinourscence and the CK-2 inverted
Olympus microscopes. Results showed that both the upper and lower epidermises
are cutinized. Stomata are found on both epidermises, which is amphistomatic, and
stomata! type is anomocytic. Trichomes are present on both epidermises and are
multicellular. Trichome length ranges from J0-20 mm in length and distributed in
clusters and rarely in isolation. These structural features of the leaf epidermises
may be responsible for the adaptability of Plumerias to different environments
especially along dusty, open, arid roadsides.

Keywords: stomata, trichomes, amphistomatic, anomocytic, multicellular,

epidermis
66 Trcms. Nat. Acad. Sci. & Tech (Philippines) Vol. 28 (No. I)

BSD No. 18
INDUCTION OF CALLUS IN LEAF EXPLANTS OF lAgmtroemia speciflsa (L.)
Pers. AND DETECTION OFCOROSOUC ACID

Maria Reichcnber C. Arcilla 1, Lilian B. Ungson.i..1,

and Almario D.J. Baltazar'

1
Faculty ofMedicine and Surgery, University ofSto. Tomas Espana St.. Manila
1
lnstitute of Biology, College of Science and
1
Natural Sciences Research Institute, University of the Philippines Diliman
~usE TECHNO Corp. l l-30 Mekong SL Riverside Sulxl. Ange!es City Pampanga
*Email: !Hian.ungson@up.edu.ph

Lagersrroemia speciosa ( L.) Pers. {Family Lylhraceae) is a dedduous tree

that grows in th.e Philippines, India, and other subtropical and tropical countries.
This species, commonly known as crape-myrtle and locally cal led "banaba" in the
Philippines. is known to have hypoglycemic effects. Corosolic acid is among its
active components. This study aimed to induce callus development in leaf explants
in vitro and to detect presence and concentration of coroso\ic acid.
Leaf ex plants from one-year old seedlings were treated with ascorbic acid to
prevent browning prior to surface sterilization with. 70% ethano ! (EtOH) and 20%
sodium hypochlorite (NaOCI). Leafexplants were grown in Murashige and Skoog
(MS) medium supplemented with naphthalene acetic acid (NAA) and kinetin (KIN) at
combinationsof5 iM: and 20 iM ofNAAand 0 lM, 1 iM, 5 iM.. and20 iM of KIN. Ten
exp!ants were used per treatment. Data were collected 011 abundance of callus
pinheads l cal li <I mm dia.) and clumps {call i e'" l mm dia.).
Calli developed on the exp\ants. espec iall~ along the margins, seven days
after inoculation except at honnonal combination of 5 i..\I ~A..\: 0 i.\1 KJN. Treatment
with 20 IM NAA: 5 iM KlN ratio produced the highest mean number of pinheads
( 1.89) while that with equal concenrra!ions of~A. .A and Kf.' 120 i\UO lM rreaonent)
produced the highest mean number ofclumps I l .60) rouneen days after inoculation,
largest mean size of the clumps (2.46 mm) was also obsen ed in the 20 1M NAA: 20
lM KIN treatment. After 53 days m culture. 20 l\1 ~AA: \ i~ KIN treaLment
produced the largest amount of call i in terms of fresh \\ e1g.h1 (ca 3DO mg).
Approximately 50 mg of oven-dried cam per rreaonent \\ere subjected to corosolic
acid analysis by high performance liquid chromatograph~ \HPLC). Percent yield of
corosolic acid found in calli of all treatments ranged from 0. 20 ro 0. 39%: the highest
value was in 5 JM NAA: 0 lM KIN treatment.

Keywords: Lagerstroem ia specios11. callus, coroso! ic acid.. narhthalene acetic acid

(NAA), kinetin (KIN)
 Trans. Nat. Acad. Sci. & Tech (Philippines) Vol. 28 (No. 1) 67

BSDNo.19
PRErARATIONANDCRYOPRESERVATIONOFCYTOPLASTSFORTHE
"PRODUCTION OF BOVINE AND BUBALINE CWNALEMBRYOS

Edwin C. Ata bay*, Eufrocina P. Atabay, Felomino V. Mamuad and Libertado C.

Cruz

Philippine Carabao Center, Central Luzon State University,

Science City of Munoz, Nueva Ecija
Email: atabay22@yahoo.com

One major factor influencing success in cloning by nuclear transfer (NT) is

the recipient oocyte. The development of efficient procedure for preparation and
utilization of in-vitro matured oocytes as recipients for NT is therefore essential.
This study was conducted to: prepare cytoplasts with high enucleation rate and
developmental competence, determine the effects of activation treatments, and
utilization of vitrified bovine oocytes as recipients for intraspecies and intergeneric
somatic cell NT. In study 1, vortexing cumulus-oocyte complexes in hyperosmotic
media for a long period moved the first polar body and reduced enucleation rate.
Removal of 20% cytoplasmic volume increased enucleation rate and showed no
deleterious effect on subsequent development of reconstructed bovine embryos.
In study 2, NT embryos were constructed with bovine oocytes of 3 different
preparations: enucleated oocytes treated with calcium ionophore for 5 min and
cycloheximide for 5 h; treated with ethanol for 7 min and cycloheximide for 2 h, and
those without treatment. NT embryos constructed with ethanol-treated oocytes
showed similar rates of fusion, cleavage and blastocyst formation to those of the
non-treated oocytes. NT embryos constructed with ionophore-treated oocytes
did not show any pronucleus-like structure and showed lower cleavage rate and
no development to blastocysts. In study 3, in-vitro matured bovine oocytes were
vitrified in microdrops. After wanning, around 90% of the vitrified oocytes were
morphologically normal. The enucleation rate and development to blastocysts
were similar to those constructed with fresh oocytes. Intergeneric NT embryos
had lower cell numbers than the intraspecies NT blasocysts. In conclusion,
aspiration of the first polar body and adjacent 20% cytoplasmic volume after
removing cumulus cells by vortexing for a short period using hypo-osmotic media
increased enucleation rate of bovine oocytes. This study also demonstrated the
efficacy of microdrop vitrification procedure and the successful production of
buffalo clonal embryos using vitrified bovine oocytes.

Keywords: Activation, bovine oocytes, enucleation, intergeneric, vitrification

68 Tram. Nat. Acad. Sci. & Tech (Philipplri~s) ~bl. 28 (No I)

BSDNo.lO
VITRITICATIONANDINVITROCULTIJREOFMOUSE PREANTRAL
FOLLICLE'S: A MODELFORMAXIMUMlJITI.JZATJON OF
OVARIAN FOLLICLES IN WATER BUFFAI.OES

Eufrocioa P. Atabay*,1 Edwin C. Alabay' and Libertado C. Cruz1

1Philipppine Carabao Center, National Headquarters and Genepool

Science City ofMunoz, Nueva Edja
Email: bingay2003@yahoo.com

In-vitro culture and cryopreservation of ovarian pre-antral fo\lic les is a new

tool for rescuing genetic materials in mammalian ovary for offspring production.
The present study demonstrated successful production of pups from vitrified pre-
antral follicles in mice which would then be applied in buffalo species. Preantral
follicles mechanically isolated from ovaries of 12 day old mice were exposed to 2
mol ethylene glyco 1for 2 or 5 min and then to vitrification solution ( VSJ containing
6 mo! ethylene glycol and 0.3 mol raffrnose for0.5, 1.0. or 2.0 min before vitrification.
The vitrified and fresh preantral folHc\es were treated with collagenase, and the
oocyte-granu\osa cell complexes (OGCs) obtained were cultured in-vitro for 10
days in membrane inserts. Preanrral follicles exposed lo 2 mol ethylene glycol for
5 min then to VS for 0.5 or 1.0 min snowed highest surv1 val rates after warming. The
follicular loss ofter warming was approximately 20%, After in-vitro culture, the
proportion of vi ab le OGCs from vitrified follicles was I 0'% lower th.an that of fresh.
preantra\ follicles. Th.ere were no differences in the rates of maturation, fenilization
and development to blastocysts between tne oocytes derived from vitrified follicles
and those from fresh preamral follicles; however, the developmental competence
of oocytes derived from both vitrified nnd fresh preantral follicles grown in-vitro
was lower than that of oocytes grown in-vivo. One of the recipient mice th.at
received 20 blastocysts derived from vitrified preantral follicles gave birth 10 six
live pups. The ability to rescue pre antral follicles and produce offspring is of great
bene Ht in buffaloes because of the inherently low number of preantral follicles in
their ovary compared to cuttle. The vitrification and culture of preantral follicles is
therefore a potentially valuable reproductive technology as this will allow maximum
utilization of the limited number of ovarian follicles in water buffaloes.

Keywords: Buffnloes, preantral follicles, vitrification, mouse, ethylene glycol, in

vitro culture
 Trans. Nat. Arad. Sci. & Tech. /Philipp111es; Joi. 28 (J;'o. /; 69

BSD No. 21
AN INfTIALJNVESTIGATION ON1HE MORPHOLOGY,
ULTRASTRUCIUREAND DEVELOPMENTOFTHE ElASMOIDSCALES
OFMULLETFISHES(PERCIFORMES: MUGll.JDAE) WfIBEMPHASlSON
LUDONG (CESTRAEUS PL/CAT/LIS VAL. 1836)

Pacifico Eric E. Calderon, Marivic A. Leon, Anna Cristina V. Suarez,

Ma. Bida Olivia S. Tamayo, Julie Ann M. Valen era and Emerita C. Simeon

Department ofBio\ogical Sciences, College of Science

University of Santo Tomas Espafta Blvd., Manila
Email: creeknipacey@yahoo.com

Comparative morphologic and molecular characters of adul! mullet fishes in

the Philippines were investigated, with emphasis given on fodong (Cesrraeus
pl iwtilis Valenciennes l 836), a rare, catadromous species suspected to be endemic
to the Ca gay an River and its tributaries. Lu dong commands a price of about Php
3000 per kilogram for its unique palatability, aroma and seasonality. Although
ludong has been pronounced endangered by the Bureau offisheries and Aquatic
Resources, its biology and taxonomy are, however. yet to be fully established.
This study was taken as an initial investigation on C. plicatilis and effort was
made to present its unique morphology for its proper identification, with emphasis
on the histology and ultrastructure of Hie scale ornamentation pattern. First, five
(5) mullet species were identified by morphological characterization. Secondly, the
histology and ultrastructure of scale ornamentation pattern were invi:stigated by
combining ocular inspection, eosin· Y staining, light and scanning electron
microscopy (SEM). Finally, the possible role of fibroblast grawthjactor 8 (fgf-8)
in scale development was investigated by genomic DNA isolation, gene
amplification by polymerase chain reaction, direct DNA sequencing, sequence
alignment and phylogenetic tree construction. In conclusion, mullet fishes were
shown ta exhibit significant variations in the sub-oral regions and in the squamation
patterns. among ather morphometric and meristic data. Intra-species regularity
and inter-species variation of squamation pattern, scale morphology and
ultrastructure were obsenied. Epithelial microridges were observed on the outer
surface of the scale epidermis, forming a species-specific ornamentation pattern.
The discreteness between morphologic and molecular data suggests the existence
of both genetic cascades and epigenetic factors in scale organogenesis and scale
ornamentation development The observed scale ornamentation pattern may be
employed to further analyze phylogenie~ and infer modes of aquatic adaptation
among mu] lets. Descriptive data obtained in this baseline study are deemed essential
for further studies on ludrmg conservation.

Keywords: Cestraem phcatilis, catadromous, conservation, e!asmoid scale,

endemic, scale ornamentation, meristic, morphometric, SEM.fgf-8, ultrastructure
70 Tram· Nat Acad Sci_ & Tech (Pkilfpp1ries) Val. 28 f!l'o /J

BSDNo.22 ,
THE FRESHWATERANDSEMl-TERRESTRIALBRACHYURANSOF
SELECTED AREAS IN LUWN ISLAND,PHILlPPJNES

Jimmy Tevar Masagca

Graduate School of Educ11tion. Arts and Sciences

De La Salle University Dasmariiias, Cavite 41 l 5
Email: junasagca@mail.dasma.dlsu.edu.ph

The freshwater and semi-terrestrial brachyurans of selected areas in Luzon

were studied on the basis of field collections c.arried out in 2004 to 2005 in Cavite,
Nueva Ecija, Tarlac. Camarines Sur, Albay and Catanduanes. A total of 116 crab
samples were obtained, preserved and diagnosed in the laboratory for taxonomic
characters. Eight (8) taxa are reported and described here. The crabs identified
belong to four farni!ies, namely: Potamidae Ortmann. 1896; Parathelphusidae Alcock,
I 910 (sensu Ng, 1988); Grapsidae Mcleay, 1838; and Gegarcinidae Mcleay, 183 8.
Two unidentified endemic potamid crabs are tentatively described as Ovitomon
sp. 1 from Temate, Cavite and Ovitoman sp. 2 from Sulong Falls in San Miguel,
Catanduanes. Although the specimens examined from Cavite showed striking
similarities with Ovitamon artifrons Burger, 1894, the snucture of male gonopodium
(G 1) does not resemmble that ofth.e specimens described previously from Tagayta)'
by Ng& Takeda in 1992. A new endemic species O\'itamon ca,,itensis (sp. ov,?) is
proposed here. Two riverine crabs belonging to the farni!> Parathelphusidae are
reported here as Sundathelphusa sp. I and 2 from Guimba, Nueva Ecija and
Moncada, Tarlac, respectively. Out of the three species of the widely distributed
grapsid crabs (of the genus Varuna H. Milne Edwards. 1830). two species are
reponed here as Varuna alrimana and r( literara_ Among the semi-terresrrial crabs,
Sesormops (Sesarma) impressum H. Milne Edwards, 1887 (from Lictin River, San
Andres, Catanduanes Island) and Cardisoma carnifex Herbst. l 794 (from Balayan.
Batangas) are also described in this continuing project on the "Biodiversity of
Freshwater Crabs of Luzon, Philippines" at De La Salle Univmity-Dasmarifias.

Keywords: Brachyurans, crapbs, taxonomic characters

 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. J) 71

BSDNo.23
MERISTICAND MORPHOMETRICCHARACTERS OFAN ENDEMIC
FRESHWATER CRAB, OVITAMON CAVITENSIS (SP. NOV.?)
(BRACHYURA: MALACOSTRACA)FROM CAVITE
LUWN ISLAND, PHILIPPINES

Jimmy T. Masagca 1 and Cherry Z. Cuevas2

1
College of Education, Professional Education Department-Graduate Studies
2
Biological Sciences Department, College of Science
De La Salle University-Dasmariftas
Cavite 4115, Philippines
E-mail: jtmasagca@dasma.dlsu.edu.ph

Given the paramount importance of the biodiversity in freshwater systems of

the country to generate more information on the crab fauna) diversity and the need
to conserve the remaining natural populations of these groups of crustaceans, a
research project under the research program on the Patterns of Biodiversity in
Aquatic Systems is being carried out at De La Salle University in Dasmarinas
(DLSU-D). Several crab specimens which are wholly freshwater species in three
families, Potamidae Ortmann, 1896, Parathelphusidae, Alcock, 1910 (both sensu
Ng, 1988), and Grapsidae McLeay, 1838 (partim) were collected by handpicking
and with the use of crab traps ("pukot" and "bintol") from different riverine and
lacustrine environments of southern Luzon (Cavite, Quezon, Camarines Sur, Albay,
Sorsogon). These specimens are currently being diagnosed taxonomically for new
records and possibly new species. The freshwater crab fauna of the Philippines is
poorly known, thus more systematic studies are needed since many more species
undoubtedly still await for discovery. During field collections in Cavite, specimens
of an endemic wholly freshwater potamid species (tentatively identified as Ovitamon
cavitensis (sp. nov. ?) was obtained from a forest stream in Mts. Palaypalay/Mataas
Na Gulod National Park (200 to 360 meters above sea level or asl). A total of sixty
(30 males and 30 females) were described and subjected for meristic and
morphometric analyses. Diagnostic analysis shows that carapace ovoid, branchial,
cardiac, urogastric and cervical grooves very shallow; postero-lateral regions appear
smooth; external orbital angle broadly triangular, epibranchial tooth low, but clearly
separated from external orbital angle; antero-lateral margin convex, postero-lateral
margin appears converging; male abdominal distinctly triangular, segment 7 slightly
longer than segments 6 and 5, lateral margin of segment 7 straight, lateral margin of
segment 6 slightly convex; third maxilliped exopod with well-developed flagellum,
longer than the width of the merus; ischium with sulcus, not medial; and dorsal
margins of chela not serrated, and finger slightly longer than palm. The first
gonopodium (G 1) stout, cylinder-shaped, curved towards the inside, and surfaces
72 Trans _'Vat. Acad. Sci. & T~ch. (Philippi11c5) Vol. 18 (No. 1)

covered with hairs. Meristically, 0 cavitensis (sp, nov.?) in both sexes consist of
7 abdominal segments, 2 anterolateral teeth protected by the third ma.i.illiped,
numerous well-developed inner distal spines in the cheliped and maxilliped; and
spinnules in the cho!liped and maxi!liped. Morpliometric analysis includes 7
characters (carapace length, Cal; carapace width, CaW; maxil\iped length, Mal;
cheliped length, ChL ); propodus length, Prl; frontal margin. FrM; and eye diameter,
E_vD). Results indicate an observed difference between male and female samples
(as to Cal, Mal and FrM} of the endemic crab under study (O. cavitensis sp. nov.
? ), which belongs to the genus 011itarnon Ng et Takeda 1992 (Potamidae Ortnmann,
1896). Further investigations are needed, particularly on the morphology
(ultrasrructures) of the sperm transfer tubes in the male gonopodium l G1).

Keywords: Ovitamon cavitensis, Cavite, lacustrine,

BSDNo.24
MONSOONAL WINDSINFLUENCETIIEASSEMBLA.GE SJ'RUCI1JRE,
ABUNDANCEAND DISTRIBUTION OF NET PHYTOPLANKTON JN ILIGAN
BAY,NORlHE.RN MINDANAO

Rosabeth Morandarte and Ephrime B. MetilJo•

Department of Biological Sciences, Mindanao State University-11 ig1m Institute

of Technology, A. Bonifacio Ave., tligan City 92 00
Telephone: (063) 221 4050 up to 55 local 13 7
E-mail: ovcre-ebm@sulat.msuiit.edu. ph

The coastal and oceanic waters of the Philippines are mainly exposed to two
dominant wind patterns annually, the north easterlies during the northeast (NE)
monsoon and the south westerlies during the southwest lSWJ monsoon. We
studied the structure, abundance and distribution of surface layer net
phytoplankton assemblages in Jligan Bay during the southwest and northeast
monsoons. Vertical samples were co\!ected at 24-station sampling grid on July-
August WOO (SW monsoon) and November-December 2000 (NE monsoon). The
abundance of each phytoplankton Genus was estimated following the Dtermohl
method. Assemblage srructure and distribution were analyzed using multivariate
and univariate routines available in the PRIMER sofh.vare (Warwick and Clarke
2001). Abundance was significantly higher in the NE monsoon. Although
assemblages in the two monsoons have similar generic richness, cyanobacteria
(Trichodesmium) dominated the NE monsoon assemblages while diatoms
(Chaetoceros) in the SW monsoon. Bray-Curtis similarity dendrograms and
multidimensional scaling plots revealed a bay-wide contour of assemblages that
 Tr.ms. Not. Acm:/. Sci. & Tech !Philippines) Vol, 28 (/l'o, 1J 73

have high, moderate, and low levels in generic diversity and relative abundance.
All three levels were observed in the SW monsoon assemblages, but only two
thigh and low) in lhe NE monsoon samples. High level stations are found in the
southwestern sector of the bay, the low levels near mouths of major river systems,
and lhe moderate level in other locations. Factors including salinity, temperature,
and total dissolved phosphate weakly explain the spatial variations observed,
thus conrrasting wind patterns and other hydrodynamic processes during lhe two
monsoons are also important in structuring phytoplankton assemblages in lligan
Bay.

Keywords: phytoplankton ecology, Trichodesmium, Chae1aceras, northeast

monsoon, southwest monsoon, multivariate analysis, lligan Bay

BSD No. 25
MODULATION OF ANGIOGENESISINTHECHICKEMBRYO
CHORIO.ALLANTOIC MEMBRANE(CAM) BYTHE POLARAND
FRACTIONAL POLYSACCHARIDE EXTRACTS OFGanoderma applanaJum

Mikhael Cereno M. Bajo 1, Lydia M. Bajo2, Roberto M. Malaluani

and Franco G Teves111

1
Molecular Genetics and Microbial Technology Labora1ory,
Department of Biological Sciences,
1 Department of Chemistry, College of Science and Mathematics

'Department of Chemical Engineering Technology,

Schoo 1of Engineering Technology
MSU-fligan Institute ofTechnology, 9200 I!igan City
*Email: franco teves@yahoo.com

Ability to regulate angiogenesis, or de novo formation of blood capillaries,

finds its significant role both in physiological and pathological conditions. The
rreatment of certain neoplastic cases with extracts of Ganaderma sp. has been
documented in China and other Asian countries, although the specific mechanism
rs still not very clear.
A polar fraction and four fractional polysaccharide extracts from Garwderma
applanatum were tested for angiogenesis modulating effects using the chick
embryo chorioallantoic membrane (CAM) assay, One-day old fertilized chicken
eggs were treated with three concentrations of the G applanamm extracts in three
replicates. One square centimeter "windows'' were aseptically created, by removing
shell materials within specified areas. Two to three milliliters ofalbumen was removed
using sterile pipettes to expose the CAM, which was then treated with O. l ml of the
74 Trmu. Nat_ Acad Sci & Tech_ (Philippines/ Vol 28 (No Ii

extracts. The "windows" were sealed with 16-ply sterile gauze taped onto th.e shell
and the eggs incubated at 37"C until the seventh day. Semi-quantitative scoring
was done by removing shell materials from the periphery of the "windows'" and
comparing the degree ofvascularillltion with the control.
The polar !Taction exhibited a significant angiogenic stimulation whereas
three out of four fractional polysaccharide extracts showed significant inhibition
of angiogenesis as analyzed using Scheffe's post hoc test and t-test. These
differential effects seem to demonstrate the presence of more than one type of
bioactive components in G applanutum. His therefore possible chat one mechanism
for the anti-neoplastic effect of this shelf fungus' extract is tllrough angiogenesis
modulation. Tumors cannot thrive without abundant blood supply.

Keywords: Angiogenesis, chorioal\antoic membrHile, polysaccharide, Gunoderma

applanalllm

BSD No.16
GEOMETRIC MORPHOMETRICAPPROACHES10THE. DISCRIMINATION
OFSTOCK POPULATIONS OF FRESHWATER COMMERCIAL FJSHES
FROM BALOI LAKE IN LANAO DEL SUR

NashebaATumpac, Mark Anthony J, Torres and CesarG. Demayo

Depanment of Biological Sciences

College of Science and Mathematics
MSU-Iligan Institute ofTechnology, lligan City
Email; c_gdemayo@yahoo.com

Th.e truss network in fishes has been used as a fishcrie5 management tool
efiec\\\'e )n \he 6.t!l.t.T\}l\\l\lt\ !\It morv\\oll\llbica\ variation and identification Of stocks,
thus improving the biological basis of management. Traditionally, this is analyzed
and compared by collecting a series of measurements between landmarks that
form a regular pattern of connected quadrilalera!s or cells across the body form
using a vernier caliper. In this paper, however, the truss networks in five fresh water
commercial fishes are analyzed using the methods of geometric morpbometrics,
which includes the generation and analysis of thin-plate spline transformation
grids, partial warp scores and interlandmark distaoces. This study aimed to present
an alternative computerized approach in the collection and multi variate analysis of
fish truss network morphometric data.
A. total of two hundred forty.four (244) individuals were sampled. Sample
sizes for each species are as follows: Glossogobim sp. (n = 40) , Hypseolotris
agilis (70), Anubas tesrudineus (42), Puntius /uniba (69), and Cyptinus curpio
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I) 75

carpio (23). These fishes were scanned at 600 dpi. Then, a total offifteen landmarks
that are distinctive and homologous from specimen to specimen were selected
around the outline of the fish form. The X-Y coordinate value for the positions of
landmarks is digitized for each fish with the use of the Scionfmage image digitizing
and processing software. The X-Y coordinate data is then transfonned into linear
distances by computer using the Euclidean Distance Matrix Algorithm (EDMA).
This transfonned data was then used as morphometric variable for multivariate
methods of statistical analysis. Such as principal component analysis, discriminant
function analysis and cluster analysis.
The original coordinate data were also used to compare shape differences
among the different species. Because, all samples for all populations were taken
from different ages, there is a need to eliminate the size effect in the data set. This
was done by subjecting the coordinate data to procrustes fitting. Then, the
infonnation contained in the procrustes-fitted coordinates were used to reconstruct
the truss networks of individual fish samples using thin-plate spline transfonnation
grids. Morphological variations within and among the different species were
summarized using PCA and other multivariate methods of statistical analysis. Global
and localized variations were also determined through the analysis of partial warps.
This study showed variations in the sizes and shapes of the individuals
within and among the different species as evident from the thin-plate spline grids
and supported by the results of the PCA and dendrogram generated through
Cluster Analysis. Discriminant analysis showed that the identification accuracy
was between 93% and 100%, and global identification accuracy was 98.6%.
Morphological differentiation was shown to vary between the sexes in all fish
species as a result of sexual dimorphism. This suggests that females and males
should be treated separately in truss network analyses to remove the effect of sex
from the result. The results of this study proved the usefulness of geometric
morphometric approaches to the study stock populations of freshwater fishes.
which are powerful in reconstructing and comparing the truss networks of fishes.

Keywords: truss network, geometric morphometrics, cluster analysis, discriminant

analysis
76 frmrs. ,-,.,·at. Acad. Sci, & Tech. (Philippmes) Vol. 18 fNo. IJ

BSD No. 27
BIO LOG V OF Eocanth~conafurceUata (Wolff) AND ITS PREDATORY
CAPACITY AGAINST CORN BORER (Ostrilliafumacalis)

Somkid Nuchphun and Teotimo M. Agaaoo*

Plant Protection Service Division, Department of Agricultural Extension,

Bangkok, Thailand and Central Luzon State University
Science City of Mu~o:z, Nueva Ecija, Philippines
Email: tmaganon@lycos.com

The biology of Evcanthecon. furce/lata was studied under laboratory

conditions. The insect oviposition period ranged from 20- 29 days with an average
of 23 .4 + 3 .65 7. The female laid eggs ranging from 7 to 104 (X "' 72.88 + 25. l l ).
The egg incubation period was 5.20 + 0.837 days. The nymph molted five
times with a total nympha! stage days of 18. 84 ... 2 .160 broken down as: I '1 instar,
2.92 + 0.846; 2"° instar, 3.56-+ 0.501; 3n1 instar, 3.46,. 0.503; 4"' instar, 3.50 + 0.496 and
5"' i nstar, 5.40 + 0.496. The total life cycle was 24 .5 2 + I .787 days. The adult male
lived within a range of36.40 days after emergence whereas the female Jived 42.49
days after emergence.
The total predatory period from the second nymphal ins tar to death of adult
was 45.23 days.
Fuactional response of adults as predator on second and third larval instars
of 0. furnacalis increased with an increased in prey density. The same trend was
observed on the predatory capacity of the nymph al stages. Adults predatory
capacity is higher than the nymphal stages.
The results showed that E. fiucellata was not a good predator if the com
borer density is high, whereas if the com borer density is low, E. furcellata could
he a good predator.

Keyword!i: Eoconthecona jiirceliata, com borer, Ostrinia/urn11cafis. biological

control
 Trans. Nat. Acad. Sri. & Tech. (Philippirre.s) Vol. 28 r.-...'a. J ! 77

BSDNo.28
FIGH11NG BEHAV10ROFTHEPIDLIPPINE DERBY SPIDER
Neoscona punctigera (Doleschall)

Aimee Lynn A. Barrion

UPLB Museum of Natural History, Upper (Forestry) Campus

University of the Philippines Los Bail.as, College. Laguna 4031
EmaH: aranea95@yahoo.com

Filipinos are game-loving people. They have managed to utilize an assortment

of animals in their games and leisures like cockfighting, horse racing. dog racing,
carabao fighting and spider derby. In most spider games, females of the species
Neoscona pu.nctigera (Doleschal\) appear to be commonly used. However, the
predatory nature of this spider is yet to be explored. Thus. this study provides
preliminary documentation and explanations for spider fighting behavior. A wooden
I-shaped rod with a catch at the bottom was used as a fighting arena. Spiders
whose body sizes ranged from 13 - 15 mm were grouped as the large mature
females while those whose body sires ranged from I 0 - I 2.5 mm were designated
as small mature females. Reproductive status was detennined by observing the
epigynum. Oviposited spiders were allowed to fight 2 days after oviposttion.
Complete sequential steps of the fights were observed, described and recorded in
video. Details of the spider matches were presented in kinematic graphs. Matches
in valving spiders of different sizes showed that larger spiders win in all encounters
that resulted in actual combat (55 of75 matches). Only in 21 of the 75 total matches
between two large female spiders actually resulted in an encounter. On the other
hand oviposition status did not affect lhe winning chances of the spider. In view
of these observations. two hypotheses on the origins of fighting behavior of
derby spiders were proposed. One is that the aggressive behavior of derby spiders
is due to maternal care, while the other is linked to territoriality.

Keywords: behavior, derby, epigynum, kinematic graphs, Neoscona punctigera.

(Doleschall), matches, oviposition. spider
78 Trans. Var Acad Sci. & Tech (Philippi11es,1 Vo/_ 28 (!l'o. J)

BSDNo.29
ALTERNATE HOSTS OF CORN EARWORM, Helicoverpa amzigera (HB.)
AND CUTWORM, Spodoptera litura (F.) IN ONlON REFUGIAS

B.F. Cayabyabl,C.L. Padilla 1, J.R. Dea non, Jr. 1, D. Vargas, Jr.'

and R.G Bayot1

1
National Crop Protection Centi!r, Crop Protection Cluster
1
Crop Science Cluster, College of Agriculture
Universiry of the Philippines Los Bafios
College, Laguna 4031, Philippines
Email: bfcayabyab@laguna.net

This study was conducted from 2002-2005 in four towns of Nueva Edja
which are the main onion growing areas in the country. It aimed to identify alternate
host plants com earwonn, Helicoverpa armigera (Hb.) and cutwotm. Spodoptera
litura (F) are major pests of vegetables and grains in the Philippines in onion
refugias of Laur, Gabaldon, Pantabangan, and Bongabon towns.
The presence of these two pests was done through ocular observation on
the boundaries of onion fields where weeds abound. The results showed that the
following species harbor these two pest: Amarcmthus spinosus L, Stachytarpheta
famaicensis (L) Vahl., Polygonum barbatum L., Cas~·ia wra L., Melochia
corchorifolia L., and Chromolaena odorata L.
Onion farmers apply sixteen times spraying on their fields to control these
pests. Knowledge of alternate host plants or where to find these pest can Jessen
pest management cost by directing control measures on refugias where these
alternate host and these two larval pc!!! are present Hence. population build up of
these pest that infest onions is reduced.

Keywords: refugias, com earworm, cut\lr'otm, indicator plants, onion, larvae

 Tran.~ .."iat. Acad Set & Tech (Pliiltppines) l'ol. 28 (So IJ 79

BSD No.30
ON10N MAGGOT/FLY, Delia an1ip11 MEIGEN (DWfERA:
ANATHOMIXIDAE): ANEW PFSTOFONION IN THE PHILIPPINES

Bonifado f, Cayabysb1 MarioV. Navasero1, Daniel G Vargas1, Paul

Rommel Elvira 1, Rolando G Bayot', Melvin D. Ebuenga 1,
Mar Antineo1, and Michael Csbslles3

1
Crop Protection Cluster, University of the Philippines
Los Bal'los, College, Laguna
1
Office of the Provincial Agriculturist Lingayen Pangasinan
1
A\lied Botanical Corporation, Quezon City
Email: bfcayabyab@laguna.net

Since 200 I to date onion farmers in Pangasinan complained of damages in

their onions described as root rot of seedlings and bulb rot The onion leaves
become brown and eventually dry up.
Field collected damaged bulbs from Amancosiling, Bayambang, Pangasinan
were brought to the Plant Quarantine Suppon Laboratory, UP Los Bafios in 2004-
2005 cropping season. Each bulb with visible larva was separated and these were
reared in the laboratory. Emerging adults were put in cyanide bonles. These
adults were pinned and stored in stwidard insect boxes. Collection of adults
through sweep net was also done in infested fields. The adults onion fly were
separated and stored in insect boxes.
The male is large (6 - 7 mm in length), with densely grey to yellow-grey dust
on thorax and abdomen. The eggs are about 1.25 mm long white and elongate-
ovoid, resembling grains of rice. The larvae are typical elongate creamy-white
maggots, tapering towards the head end and truncate at the posterior end where
rwo breathing sp irades are situated. The pupae are ovoid or seed-shaped. reddish-
brown. The posterior spiracles are situated on small bosses. Adults are slender
ye1lowish·grey flies, 6 - 7 mm in length. The signs of at1ack are yellowing and
wilting of the outer leaves. Green and apparently healthy leaves will become
flaccid, and the who le plant may collapse. Later generations of Jar11ae tunnel into
the on ion bulbs as well as attack the roars. This new pest of on ion is prevalent in
onion growing areas of Pangasinan such as Alcala. Bay am bang Basista and nearby
towns. It has also been found in Nueva Eda and Nueva Viscaya onion areas.

Keywords: Onion maggot/fly, New PesL Onion, Delia antiqua

80 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. !)

BSDNo.31
PHILIPPINE PREDATORYMITESOFTIIE GENUS CHEIROSEIUS
(ACARI: ASCIDAE)

lreneo L. Lit, Jr. *1.z and Leonila A. Corpuz-Raros2..i

1
Department ofForest Biological Sciences
College of Forestry and Natural Resources,
2
Entomology Section, Museum ofNatural History, and
1
Crop Protection Cluster,
College of Agriculture, University of the Philippines Los Bafios
College, Laguna 4031, Philippines; *
Email: illitjr@yahoo.com

A taxonomic study of Philippine mites belonging to the genus Cheiroseius

(Acari: Ascidae) was conducted. This is a group consisting of predators of soil
arthropods and other small animals like nematodes and are, therefore, potential
biological control agents against soil-inhabiting pests. Habitat-wise, they can be
found in soil, decaying plant litter and mosses. From only three previously reported
species, namely: Cheiroseius curtipes (Halbert), C. nepalensis (Evans & Hyatt)
and C. serratus (Halbert), there are now 11 species, including four described as
new to science and four new fauna! records. Two of the new species are from
Mount Banahaw de Lucban, coded Cheiroseius spp.nov. BDL l & BDL2, and the
two others are from Polillo island, coded Cheiroseius spp.nov. POL 1 & POL2.
BDLI is unique in having a prominent dorsal shield, so produced as to form a
"humpback" effect, whereas BDL2 has very unusual ornate genital shield. POL I
has intricate reticulate patterns on the dorsal shield and POL2 has scale-like patterns
on the sternal and genital shields. The four new records are C. browningi (Evans
& Hyatt), C. cassiteridum (Evans & Hyatt), C. phalangioides (Evans & Hyatt)
and C. politulus Tseng. A key to Philippine species, descriptions and illustrations
are provided.

Keywords: Ascidae, Acari, Cheiroseius, mites, predatory mites, soil-litter fauna

 Trans. Nat. Arad. Sci. & frd1. (Philippines/ Vol. 28 fNo. /1 SJ

BSD No. 32
THE PHILIPPINE ENDEMIC STICK-INSECT GENUS OBRIMUS STAi.i 1875
(PHASMATODEA:HETEROPIERYGIDAE:OBRIMlNI)

I reneo L. Lit, Jr. "' 1.l and Orlando L. Eusebio'

1Department of Forest Biological Sciences

College of Forestry and Natural Resources

2
Entomology Section, UPLB Museum ofNatural History
University of the Philippines Los Bafios
College, Laguna 4031, Philippines:
•E-mail: illitjr@yahoo.com

Studies on Philippine stick insects have recently gained considerable interest

from many parts of the world, especially with the recent tide of discoveries of new
species and genera. Among Philippine stick insects, the tribe Obrimini stands out
as being endemic at a suprageneric level. As a contribution to the systematics of
th is group and to the documentation of Philippine terrestrial arthropod biodiversity,
a talwnomic review of the genus Obrimus St~l. 1875 was conducted. Six species,
including two possibly new ones, are described and illustrated. They are Ohrimus
bicofanus Rehn & Rehn (lsarog), Obrimus bufo (Westwood), Obrimus sp.1 ex
Northern Sierra Madre, Obrimus sp. 2 ex Southern Luzon, Obrimus mesoplatus
(Westwood) and Obrimus uicha.ncoi Rehn & Rehn (Greater Sipit Watershed, Mt.
Makiling). The differences of these species lie in the partem of their annature
which consists mainly of spines and tubercles. All species are of narrow endemic
distribution and may be possible candidates for listing as threatened or vulnerable
species, considering the rapid destruction of forest habitats and their current
status as among favorites of hobby collectors in Europe and other advanced
countries.

Keywords: Heteroptcrygidae, Obrimini. Ohrinms. Phasmatodea, Philippine

endemic arthropods, stick insects. terrestrial arthropod biodiversity
82 Trans. _l<,'a/_ Acad Sci & Tech. !Philippines) Vi.ii 28 IXo I!

BSD Nu. 33
BACTER1AL INHIBITION OF Aspergil/,usjltJvus GROWTH AND
AFL.A TOXIN BIOSY NTHESIS: Bacillus amylollquifacien.r ET20B4, .~
POTENTIALBIOCONTROLA.GENT

Sharon May D. Erasquin, Franco G Teves*, Ermelinda G Tobias

and Faith 8.Amorado

Molecular Genetics and Microbial Technology Laboratory

Department ofBiological Sciences, CSM
MSU-Iligan Institute ofTechnology. 9200 lligan Cit}
•Email: fr an co_ teves@yahoo.com

Damage to crops by fungal agents like Aspergillus jlcn•us and Aspergillus

parasitic11s not only lower their yield and quality but also continue to pose health
hazards to consumers as a consequence of aflatoxin production. While genetic
modification bestows resistance against pathogenic fungi, the use of appropriate
biological control agents may also provide an equally effective alternative.
Five pre-selected bacterial isolates (Staphylococcus epidermidis,
Agrobacrerium radiobac.ter, Bacillus megaterium, £nterob11cter aerogen.es, and
Bacillus amyloliquifaciens ET2004) were screened for inhibition of growth in
Aspergillus .flcwus and/or inhibition of aflatoxin biosynthe~is. Replicated co-
cultures on potato dextrose agar (PDA) plates. which supported the growth of the
fungus and bacteria, were prepared by parallel sn-eaking. The plates were incubated
at 30 °C and examined for fungal overgrowth on the bacterial streak after 48 h.
Inhibition of aflatox in biosynthesis was determined by co-cu \tures in potato
dextrose broth (PDB). One milliliter of l O" to lo· bacterial cells/ml and 56 ml of
sterile water-suspended A. flavus conidia ( Jff' to Io~.'m!), were: mixed in four mil!iliters
of PDB and incubated with shaking initially at 37 "C for da~ one and at J 0 "C for the
next six days. The cultures were centrifuged at 3,000 rpm for five min. The
supematants were col!ected aseptically and analyzed for aflatoxin content using
Competitive-Direct Enzyme Linked lmmunosorbent Assay (CD-ELISA) with the
VeratoxO System (Glenwood Technologies).
Results show significant fungal growth inhibition exhibited by Bffcillus
amylo/iquifaciens ET2004, but not by the other bacteria\ isolates. Interestingly, no
aflatoxin was detected in the latter tubes although substantive gtO\~th of A. flavus
was evident. Growth inhibition may be attributed to the production of subtil isin by
B. amylollquifacien.s which is known to degrade proteins extracellularly. Presence
of fungal growth without the concomitant production of afiato.\ lI1 ma) indicate the
synthesis of cell products that specifically inhibit the aflatoxin biosynthetic
pathway, but not growth.
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No J) 83

B. amyloliquifaciens is not known to exhibit pathogenicity or toxigenicity,

and is therefore a very promising candidate for the biocontrol of fungal invasion of
crops. Pending verification, the other pre-selected bacterial isolates may be utilized
for targeting only aflatoxigenic fungi, while leaving unaffected other fungi that
may play important ecological roles.

Keywords: Aspergillus jlavus, Bacillus amyloliquifaciens ET2004, aflatoxin,

subtilisin, biocontrol

BSDNo.34
MITOCHONDRIALDNAANALYSIS OF GENETIC INTROGRESSJONIN
SELECTED PHILIPPINECATILEPOPULATIONS

Genevieve Mae B. Aquino 1*, Rita P. Laude 2, Jianlin Han 3,

and Olivier Hanotte3
1
Animal & Dairy Sciences Cluster, College of Agriculture, and
2
Institute ofBiological Sciences, University of the Philippines
Los Banos, College, Laguna, Philippines
3
International Livestock Research Institute, Nairobi, Kenya
Email: gm.aquino@gmail.com

For the future improvement of beef cattle breeding programs in smallholder

farming systems, mitochondrial DNA (mtDNA) displacement loop (D-loop)
sequence variation was examined to supplement existing basic information on the
genetic composition of Philippine Native cattle. To evaluate the degree of genetic
introgression in native cattle populations representing the Ilocos, Batangas, Ilo-
ilo and Philippine Native types, a fragment of the hypervariable region in the
mitochondrial D-loop was amplified and sequenced from a total of 100 individuals
used in a previous study on autosomal microsatellite variation. Multiple sequence
alignment with published D-loop sequences of foreign purebreds and subsequent
phylogenetic analysis revealed three major clades of zebu (Bos indicus), taurine
(B. taurus) and banteng (B. banteng) maternal ancestry. Individual sequences did
not cluster significantly into geographic groups, providing evidence of migration
between populations though trade relationships between these regions. However,
haplotypes ofNative cattle were distinct from those of the foreign breeds, implying
the evolution of the Philippine populations from their foreign ancestral populations.
Patterns of mitochondrial DNA variation indicate that the Ilo-ilo type was oftaurine
ancestry with zebu genetic introgression, and the Philippine-Bali type was of zebu
ancestry with banteng introgression. The hybrid zebu-taurine composition of
mtDNA from the Ilocos and Batangas populations confirmed the results of previous
fl4 Trans. Nar Acad. Sci. & Trch (Philippines) Vol. 18 (No I)

autosomal microsatellite analysis. Molecular characterization based on combined

mitochondrial and microsatellite analysis provides a clearer perspective on the
genetic composition of Philippine cart le populations and showed the potential of
existing populations in the geograph.ically-isolated islands of the Visayas for future
genetic conservation and breed development programs.

Keywords: cattle, mitochondria! DNA, D-\oop, genetic diversity, Philippine Native

breed

BSD No. 35
SEQUENCEANALYSISAND CHARACTERIZATION OF
THE MOVEMENT PROTEIN-ENCODING COMPONENT OF ABACA
BUNCH\' TOP NANO\!IRUS(ABTV)

Vermando M. Aquino"and Maria Annila D. Ruiz

National Institute of Molecular Biology and Biotechnology

University ofth.e Philippines DilimM, Quezon City
Email: vermando.aquino@up.edu. ph

Abaca bunchy top nanovirus (ABTV) is the most important viral pathogen
of abaca. The virus causes stunting and significant reduction in yield. In this
study, the DNA component ofabaca bunchy top nanovirus (ABTV) coding for the
movement pro!ein was isolated and characurized. Oligonucleotide primers based
on published sequence of a re lated virus, banana bunchy top virus (BB TV). were
used for PCR amplifications. Tota! DNA from infected leaves was extracted from
different ABTV isolates using a modified CTAB extraction method. A 380 bp
fragment was amplified, cloned and sequenced. Sequence analysis showed th.at
the movement protein gene includes regions that codes For hydrophobic protein.
The nucleotide sequences were compared with published BBTV sequences and
showed 94 to 96% nucleotide identities with isolates from Australia and Asian
countries.

Keywords: abaca, movement protein gene. abaca bunchy top naoovirus

 Trans. Xat. Acad. Sci. & Tech. (Philippmcs/ Vol. 18 ("/<,'o_ Ii 85

BSDNo.J6
TIIE RELATIONSHIPBETWEENABACA BUNCHYTOPNANOVIRUS
(ABTV)AND BANANA BUNCHYTOPNANOVIRUS(BBTV) BASED ON
SEQUENCE ANALYSISOFDNA-3AND DNA-4 COMPONENTS

Vermando M. Aquino·and Maria Armila D. Ruiz

National Institute of Molecular Biology and Biotechnolog,v

University of the Pb ilippines
Diliman, Quezon City
Email: vermando.aquino@up.edu.ph

Bunchy top is the most devastating disea.'ie of both abaca and banana caused
by abaca bunchy top nanovirus (ABTV) and banana bunchy top nanovirns (BBTV)
respectively. ABTV is assumed to be synonymous to 1he bunchy top virus that
infects banana but this has never been confirmed. BBTV is considered to be a
variable virus with strain-complexity. Previous reports on transmission experiments
showed dissimilarity between these two viruses, although they exhibited strong
serological relationship. In this study, a comparative analysis was made on the
nucleotide sequences ofDNA-3 and ON A-4 components of both BBTV and ABTV.
fhese two DNA components contain the majorORFs encoding for the coat protein
and movement protein. Primers targeting a 586 bp gene fragment of the DNA-3
component and a 380 bp gene fragment of the DNA-4 component were used for
PCR amplifications. The resulting PCR products were cloned, sequenced and
subjected to pairwise alignments. Tile mean sequence difference between BBTV
and ABTV was 4 to 7% for the DNA-3 fragment and 2 to 4% for I.he DNA-4
fragment. Comparisons were also made for specific regions of the ORF and the
encoded amino acids. The low sequence heterogeneity obtained for the two major
ORFs suggests that these two viruses are strains or biotypes and not distinct
Nanovirus species.

Keywords: abaca, banana, abaca bunchy top nanovirus. banana bunchy top
nanovirus
86 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. ])

BSDNo.37
SHELL SHAPE CHANGES IN NINE SPECIES OF MARINE, FRESHWATER
AND LAND SNAILS USING SUPERIMPOSITION AND THIN-PLATE SPLINE
ANALYSIS OF LANDMARKS IN DIGITIZED IMAGES OF SHELLS

Rizalyn T. Borra and Cesar G Demayo

Department of Biological Sciences, College of Science and Mathematics
MSU-Iligan Institute ofTechnology, Iligan City
Email: c_gdemayo@yahoo.com

Morphometrics involves the quantitative study of form. Form is intuitively

understood that is consisting of size and shape which are never biologically
independent but are instead inextricably interrelated. In this study continuous
data was obtained to assess variations in the form of the shell of the snail. This
was determined by employing geometric Morphometrics, a method used to obtain
detailed shape information. In general, geometric morphometric methods provide
greater power than the traditional methods because the position of the landmarks
can be retained and can be graphically reconstructed. Meaning, it preserves
geometry of object studied and it allows visualization of shape differences between
specimens and between group means in specimen shape thus was used in the
study. Shell shapes of a marine snail Marmorostoma chrysostomum, freshwater
snails namely Pila polita, Belamaya angularis, P pomacea, Pseudosuccinae
peregrina and an unknown species (designated as "unknown sp. e") and three
species ofland snails namely Achatinafulica and hvo unknown species (designated
unknowns band c) were used since the shell of this group is either spherical or
heliciform or elongate ovate having three to five sutures with wide oval or circular
aperture. It has no siphonal canal and the outer lip of the aperture is not reflected.
These nine species have twenty-one (21) homologous landmarks identified thus
were the bases for their use in this study. lntraspecies and between species shape
variations were examined using procrustes superimposition and thin-plate spline
(TPS) analysis to examine local and global sources of variations in shapes. Results
ofprocrustes superimposition showed the landmark points in all the snails fall on
the same location and the general shape of the shell structure was determined.
Distinct differences can now be determined and variations in the deformation
between the species can be visually detected. Procrustes analysis was therefore
an important means of shape comparison because it removes or eliminates the
differences in rotation, translation and scaling of forms. After the landmark
configurations were superimposed, residuals were modeled with the Thin-Plate
Spline (TPS) transforming one coordinate system into another. The parameters of
the TPS transformation were used to explore patterns of shape changes between
objects. Shape changes as deformation within species of snail's shell included in
this group was made possible by fitting an interpolation function to the aligned
 I

Trans Nut. Acad. Sc1 & Tuh (Philippines) Vol. 28 (No I1 87

landmark coordinates of each specimen against the reference configuration, so

that all homologous landmarks coincide. Variations between species were observed
11s the she!ls vary in landmark points where local and global defonnations have
occurred. These can easily be observed from the graptiical presentation of the
changes in individual shapes.

Krywords: Superimposition, Thin-plate Spline Analysis. Geometric

Morphometrn·.~

BSDNo.38
SIZE AND SHAPE VARJ.ATION IN POPULATIONS OFAc/1aJinufulica AND
Amphidromw; maculiferus: A MULTIVARIATE MORPHOMETRIC
APPROACH

Ken P. Dadtda, Mark Anthony J. Torres and Cesar G. Demayo

Department of Biological Sciences, Col!ege of Science and Mathematics

MSU-lligan Institute ofTechnology, Ilig1111 City
Email: c_gdemayo@yahoo, com

Size and shape variation in selected populations of the land snai!s Achatirw
julica and A mphidromus maculiferus was determined using landmark- and distance~
based morphometric analysis. Three h!!ndred thirteen A. fii/ica and ten A.
marnliferus were sampled from Mt. Agad-agad, lligan City and from Be.langao,
Zamboanga-Sibugay. All samples were scanned at 600 dpi with the snails carefully
oriented with the aperture resting on the bed of the scanner. The two !and snail
species have obvious spiral ornamentation which allows determination of
biologically meaningful landmarks, of which twenty-two points were digitized using
the Scionlmage software. This study used distance variables based on the twenty
two landmarks with an objective of comparing the sizes of the different samples.
Distances between points were calculated using the Euclidean Distance Matrix
Algorithm. The measurements include the shell's total length, which measures the
maximum distance parallel to the axis of coiling from the shell apex to the basal
apertural edge; total width of the snail shown as the maximum distance perpendicular
to the ax is of coiling; height of the spire which is also the distance from the apex to
the aperture-suture jlUlction; distances between the included major spiral chords;
and the angulations on the comer of the aperture. These measurements give a
bal11nced overview of the shape of the snail's shell, with detailed representation of
the whorls ch11racteristic of each species. Principal Component Analysis (PCA)
was done to determine components with defined biological meanings that account
for the maximum variation of the snail samples. This multivariate method allows
numerous variables to be collected, a.nd the significant fine-scaled variation to be
88 Trans_ ,\'at Acad Sci & Teel!. 1Philippl11es) Vi1[_ 28 (}.'o. I!

extracted. Loadings for each variable in each principal component represent that
variable's contribution to the variance explained by that principle component.
Cluster analysis was also employed to group snails with similar sizes together.
Then, test for significant differences among the snails was done using the
Discriminant Function Analysis (DFA). Shape differences among the different
snail samples was detennined by subjecting the raw :.c and y coordinates of the
twenty two digitized landmarks to Procrustes fitting. This procedure eliminates
both rotational and size trans laJion al lowing for the analysis of snail shape variation
only. The Procrusted-fined coordinate values were also subjected to PCA, Cluster
analysis and DFA.
Results showed no significant size differences among the snail samples from
the two locations based on the computed interlandmark distances. A variation in
shell shapes between the two species is evident from the reconstructed images of
the snails. PCA of the Procrustes-fitted values returned a total of thirty-six
significant components with the first component explaining ninety-eight percent
(98%) of the observed variation. This variation is summarized in a scaner plot with
the Zamboanga popu \at ion shown to have broader 11pices. more expansive aperture
and wider spire base. Cluster analysis of the Procrustes-fitted values separates the
three populations into three well-defined groups. DFA of the data reveals that the
variations observed are statistically significant These results are funher discussed
in the light of ontogenetic al\ometry and parasite-induced changes in the
morphology of the snail.

Keywords: procrustes analysis, superimposition. multivariate morphomerrics

BSD No. 39.-'

HOMOSEXUAL ORIENTATION IN FILIPINO GAY MEN l]'r: RELATION TO
RELATIVE LENGTH OFlHE SECO~AND FOURTH FINGERS (2D:4D
RATIO), DEVELOPMENTAL INSTABO..ITY AA"D fRATERNAL BlRllt
ORDER

Cesar G Demayo", Mark Anthony J, Torres, Christian Mae T. Tuanzon

and Ruffy Allan V. Genilo

Depanment of Biological Sciences, College of Science and Ma thematics

MSU-lligan Instirute ofTeclmology, lligan City
Email: c___gdemayo@yahoo.com

Variations between homosexual male, heterosexual male and heterosexual

female populations were assessed based on relative lengths of the second and
fourth fingers (20:4D ratio), fraternal birth order and developmental instability.
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I) 89

Our study on homosexual and heterosexual 2D:4D ratios revealed high

heterosexual male 2D:4 D ratio than homosexual men. Comparison of the frequency
distribution of the 2D:4D ratios also showed that for most homosexuals, the ratio
is lower than of the heterosexual men. It was shown that homosexual men's ring
finger lengths were shorter than heterosexual men's. The index fingers however
show that homosexual men's index finger lengths did not differ from heterosexual
men. It is suggested from this study that there is a correlation between homosexual
orientation and the ratio of relative length of the second and fourth fingers of the
hand thus confirming previous reports.
Many other studies on sexual orientation in humans also argued to be possibly
influenced by levels of prenatal sex steroids which canalize neurodevelopment
along sex-typical (heterosexual) or sex-atypical (homosexual) lines. We measured
developmental instability by subtracting the left from the right value of the nineteen
(19) bilaterally symmetrical traits (FA) among one-hundred (100) homosexual males,
fifty (50) heterosexual males and fifty (5) females. These include digits on the right
and left hands and feet, ears and face, lengths and width of right and left hands
and feet. It is argued by many researchers that higher FA values indicate higher
incidence of developmental instability. Seven (7) fluctuating asymmetry (FA)
indices were used. The normality of the frequency distributions was evaluated
through frequency histograms that test various aspects of distributions such as
skewness, leptokurtosis and platykurtosis. Results showed variations between
males, females and homosexuals based on FA values. However, homosexual
males exhibited the highest FA values in all the indices used indicating high
developmental instability in homosexuals than heterosexual males and females
which showed low FA values.
Many earlier studies have repeatedly shown that older brothers increase the
probability of homosexuality in later-born males. The collective findings suggest
that birth order is perhaps the single most reliable demographic difference between
homosexual and heterosexual men.
We conducted a study in 2004 and 2005 involving one-hundred seventy five
( 175) homosexuals and l 00 heterosexual males assessing their fraternal birth order.
The odds and probability of homosexuality as a function ofnumber of older brothers
and attributable fraction with zero (0) ton number of older brothers were computed.
The attributable fraction, an index referring to the difference between the prevalence
of homosexuality among men with one older brother and the prevalence among
men with no older brothers calculates how much of the probability is attributable
to fraternal birth order. The results of the study showed there is an increase in the
probability of being a homosexual as the number of older brother increases. The
results of the current study confirmed earlier reports that the older the brothers a
boy has, the more likely he is to develop a homosexual orientation.

Keywords: fraternal birth order, 2D:4D ratio, developmental instability, fluctuating

asymmetry
90 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. I)

BSDNo.40
SCREENING FORAMPICILLINRESISTANCE GENES FROM VIBRIO
HARVEYINBRC 15634ANDPHILIPPINE VIBRIO ISOLATES

Prima Fe R. Franco 1•2 and Cynthia T. Hedreyda 1

1
National Institute of Molecular Biology and Biotechnology
College of Science, University of the Philippines
Dilirnan, Quezon Cityl 101
2
College of Arts and Sciences, Department of Biology
Mariano Marcos State University
Batac, 2906, Ilocos Norte;
Email: pfrfranco@digitelone.com

Vibrio harveyi, the bacterium implicated in luminous vibriosis, causes mass

mortalities in hatchery-reared larvae of the black tiger shrimp Penaeus monodon.
Antibiotics are commonly used in hatcheries and grow-out ponds as prophylactic
agents for the prevention of larval bacterial infection. V. harveyi isolates from
moribund shrimps are resistant to antibiotics. The ability of such isolates to
develop resistance to antibiotics like ampicillin was explored in this study.
Preliminary results of our studies showed that a strain of V harveyi becomes
resistant to ampicillin when transfonned with pUC 18 with ampicillin resistance.
Confirmation of the transformation process, however, showed that the genomic
DNA of the untransformed strain contains an ampicillin resistance gene. This
was done via Polymerase Chain Reaction (PCR) using primers designed based on
the ampicillin resistance genes in pUC 18. The presence of the gene in the reference
and some Philippine Vibrio isolates was observed when expected amplicons
approximately 700 bp were obtained. Optimized PCR conditions for 30 cycles
include initial denaturation at 94 °C for 5 minutes, denaturation at 94 °C for 1 min,
annealing at 57 °C for 1 min, elongation at 72 °C for 1.5 min and final elongation at
72 °C for 5 min. The PCR products were purified using NucleoSpin purification kit
and submitted for sequencing. BLAST search results show that the obtained
genes were 98% similar to the ampicillin resistance gene in pUC 18.

Keywords: Vibrio harveyi, Phillipine Vibrio isolates, ampicillin resistance genes

 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. l) 91

BSD No. 41
Relative Warp Analysis to Explore Scapular Shape, Mandible and Pelvic Girdle
in Fruit and Insectivorous Bats

Cesar G Demayo, Mark Anthony J. Torres, Angela Marie Cariaga, Gianhope

Silao, Putri Hadjariah Baser and Leonard Patrick Gaba to

Department of Biological Sciences, College of Science and Mathematics

MSU-Iligan Institute of Technology
Email: c_gdemayo@yahoo.com

One method that can be powerful in establishing structure-function

relationships is the use of relative warp analysis of digitized images. Relative
warp analysis is essentially a principal component analysis of the covariance
matrix of partial warp scores produced by thin-plate splines. The technique
produces the power of thin-plate splines to produce a visualization of morphospace
via the transfonnation grids. We elucidated the functionality of this method to
explore scapular, mandibular and pelvic girdle shapes in fruit- and insectivorous
bats. Relative warp analysis can provide both a descriptive and quantitative
description of shape. Adult specimens of bats that were accidentally caught and
injured in sampling by researchers and by fruit fanners who consider the bats as
pests in their fanns and from the museum were processed and the images generated
by digitization at 600 dpi using high resolution computer scanners. Ten ( l 0)
landmark coordinates of the mandible, eleven ( 11) from the scapula and twelve ( 12)
from the pelvic girdle were identified and transported in a processing software
providing translation, rotation, and resealing of landmarks with reference to a
specified baseline. Mean shapes for each species were detennined by averaging
shape coordinates for each landmark and these means were imported to a
paleontological statistics software developed by Hammer in 2002 for relative warp
analysis. The mean shapes for each species were compared to the reference fonn
to generate partial warps on the same partial warps. Shape differences for each
species were then revealed by examination of associated partial warps in the
transfonnation grids. A model for the defonnation of one shape to another was
then made possible by superimposition method calculating the "best fit" between
the shapes. A UPGMA (unweighted pair group method using averages) cluster
analysis was performed on the resulting matrix to explore patterns of shape in the
scapula, mandible and pelvic girdle.
Results of relative warp analysis of the mandibles of the bats show that the
evolutionary shifts in diet are reflected on the changes in mandibular shape
especially in the coronoid process and angle of the jaw. Significant differences in
shapes of the mandible between species, families and between the frugivorous
and insectivorous group were observable in these regions. Higher coronoid process
 Trans, Nat. Acnd. Sci. & Tech (Philippines) ~al. 28 (No !)

can be found in jaws of hard object diet specialist such as frugivorous bats and are
reduced in the insectivorow bats. The scapular changes within and between
groups of bats were similar and were c.:haracterized by the enlargement of the teres
major process, a landmark that serves as attachment of the teres major muscle
which is veiy important in flight. This result is expected since all the bats are
strong flyers. For the pelvic shape, results show major changes in the posterior
interior spine, dorsal margin of the crest of the i Hum, anterior margin or the ilium,
ventral margin of the crest of the ilium iliopectineal eminence, both anterior and
posterior margins of the obturator foramen. This result on the pelvic girdle
variations reflects group status in bats. The shape differences in these landmarks
also indicate the launching differences between the insectivorous and frugivorous
bats. The results of this study indicate that new methods like the re \ative warping
method in morphospace studies can be of prime irnponance in establishing
structure function relationships in living organisms.

Keywords: relative warps, frugivvres, insectivores

BSDNo.42
GENETIC ANALYSIS OF AlliumALLllNASF.S BY SEQUENCE ALIGNMENT
AND PHYLOGRAM CONSTRUCTION

Ralph Arnold S. L1tsala*, Vivian A. Panes, Vivian S. Tolentino

and Merab A. Chan

Department of Biology, Ateneo de Manila University

Loyola Heights, I l 08 Quezon City
Email: rlasala@ateneo.edu

Bioactivc compounds in garlic (Allillm sarivum) and other Af!ium species

originate from thiosulfinares such as allicin, which are produced by the aclion of
the enzyme alliinase (EC 4,4, 1.4) on cysteine derivatives, through cleavage of
carbon-sulfur bonds. ln this study, sequences of alliinases from member species
of the genus Allium were used in order to describe phylogenetic relationships
among Allium plants. Using BLAST (Basic Local Alignment Search Tool) searches
on NCBI (the USA National Center for Biotechnology Information) databases
along with searches on Map Viewer and ExPASy (Expert Protein Analysis System)
servers, alliinase and a!liinase-related sequences within the genus Alliu.m (A.
ascalonicum, A. cepa, A. chi11ense, A.jistulosum, A. giganteum, A. sativum, A.
schoenoprasum, A. ruberosum. and A. wakegi) were obtained and analyzed. By
mu !tip Ie sequence alignments using Clustal W and various distance matrix
computations, cladogrwns and phylograms were constructed to characterize the
 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. 1) 93

genetic distances among A Ilium species. Ninety nine (99) residues were found to
be identical in all sequences used in the alignment. Sixty five (65) conserved
substitutions and 43 semi-conserved substitutions were observed. The phylogram,
a branching diagram (tree) assumed to be an estimate of a phylogeny wherein
branch lengths are proportional to the amount of inferred evolutionary change,
clustered the alliinase sequences from A. fistulosum, A. giganteum, A. sativum, A.
tuberosum under one clade, while the A. cepa and A. wakegi alliinases were
clustered separately. Molecular structures of the various alliinase sequences were
also generated using Swiss-PDB Viewer or Deep View, which showed similar patterns
of the arrangement of the component modules (a homodimeric structure with each
monomer composed of the N-tenninal domain including the EGF-like domain,
followed by the central domain residues, and then the C-terrninal domain). This
study significantly contributes to our understanding of the genetic structure of
alliinase expression among members of the genus Allium. Studies on the detailed
structure of the conserved domains are required in order to elucidate further the
evolution of the alliinase gene family.

Keywords: alliinase,Allium, alignment, phylogeny, genetic distance, garlic, onion,

leek, chive

BSDNo.43
ISOLATION OF PLASMIDS FROM PHILIPPINE NAEGLERIA SP.

Fides Marie DS. Francisco• and Windell L. Rivera 1•2

1
Institute of Biology, College of Science
University of the Philippines Diliman
2
Molecular Protozoology Laboratory
Natural Sciences Research Institute,
University of the Philippines Diliman
Email: windell.rivera@up.edu.ph

Naegleria is a genus of free-living soil amoebae. One species, Naegleria

fowleri, is opportunistically pathogenic in mammals and is the etiological agent of
primary amoebic meningoencephalitis (PAM). The species is the third known
eukaryotic organism with ribosomal RNA gene (rDNA) plasmid. The rDNA structure
has been described in the strain of Naegleria gruberi; the genes carried on a 14-
kilobasepair, circular, extrachromosomal DNA plasmid. Soil and water samples were
obtained and tested for the presence of Naegleria through the enflagellation test
and polymerase chain reaction (PCR). Samples were then subjected to plasmid
extraction, and visualized using agarose gel electrophoresis (AGE). Bands with a
94 frt111J_ Nu1. ,.Jrad Sci & Tech rPhilippmesJ Vol. 28 (,Vo. IJ

size of 14-kb were observed in all samples after AGE of the extracted plasmid. This
study substantiates the presence of extrachromosomal plasmid in Philippine
Naegleria species.

Keywords: Naegferia, ribosomal DNA plasmid, en flagellation test. polymerase

chain reaction

BSDNo.44
RESTRICTION ENZYME ANALYSIS OF THE t8SSSU rDNA OF
PHILIPPINEACANTIIAMOEBA ISOLAT[S

Jenica Anne A. G a.ndionco1 and Windell L. Rivera i.z

1
Institute ofBio\ogy, College of Science
University of the Philippines Diliman
2
Mo!ecular Protozoology Laboratory
Natural Sciences Research Institute
University ofthe Philippines Diliman
Email: windelLrivera@up.edu.ph

Acanthamoeba, the most common protist found in soil, are a genus of small,
free-Jiving, and ubiquitous amoebae that exhibit a biphasic life cycle composed of
vegetative rrophozoite srage and a physiologically static cyst stage. They are the
causative agents of Acamhamoeba keratitis (AK) and granu!omatmJs amoebic
encephalitis (GAE). However, despite its medical importance, the subgenus
classification of Acamhamoeba is still problematic. Riboprinting is a promising
method for the rapid ideotification of unknown AcanJharnoeba isolates. It in valves
the use of polymerase chain reaction (PCR) couple.d with restriction fragment
length polymorphism (RFLP) of the nuclear small subunit ribosomal RNA gene
(SSU rDNA). Philippine Acamhamoeba isolate~ were collected from soil samples
obtained from different geographic locations in the country. These were analyzed
based oa morphological characteristics as well as through the restriction enzyme
analysis of I SS SS U r ON A. Majority of the isolates exhibited the group II .:yst
morphology. Data from riboprinting were analyzed using cluster analysis. Using
this method, this study was able to prove that genetic diversity exists in Philippine
A.canthamoeba isolates, but this diversity is independent of the location from
where the i8olates were obtained.

Keywords: Acanthamoeba., riboprinting, genetic diversity

 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. J) 95

BSD No. 45
LTILIZATION OFPCRFINGERPRINTINGFOR THE DISCRIMINATION
OF A PHILIPPINE HYPERVIRULENT RALSTONIASOUNACEARUM
TOMATO STRAIN

Jocelyn T. Zarate, 1Asuncin K. Raymundo* 1 and Caitilyn C. Allen 1

1
National Institute of Molecular Biology and Biotechnology (BIOTECH) and
2
Institute of Biological Science, College of Arts and Sciences,
University of the Philippines Los Banos, College, Laguna 403 l,
3
Department of Pathology, University of Wisconsin-Madison, USA
Email: joytzarate@yahoo.com ; cza@plantpathwisc.edu; akr@laguna.net

Ralstonia solanacearum is the causal agent of the serious disease known as

bacterial wilt affecting greater than 200 plant species. We are working on the
characterization of a hypervirulent R. solanacearum substrain (T73 l) that wilted
previously resistant tomato varieties compared to its wild type strain (T523 ). The
presence of additional virulence factor might impact disease management. Different
PCR- based DNA fingerprinting techniques were evaluated for differentiation of
the hypervirulent mutant strain from the wild type strain. BOX and ERlC PCR were
unable to differentiate the two strains while REP PCR and Pulse Field Gel
Electrophoresis (PFGE) generated differential bands unique to the hypervirulent
strain. Three rare cutting restriction enzymes used in generating the macrorestriction
patterns in PFGE showed that strain T523 and T73 l were very similar to each other
except for one to three differential bands. Cloning and sequencing of a 414 bp REP
PCR generated DNA fragment which was present only in the hypervirulent strain
showed that the sequence is present in the megaplasmid of the sequenced R.
solanacearum strain GMIIOOO, a tomato isolate from French, Guyana in three
different portions. The highest homology was present at the 5' side of the
transposase protein of the insertion element l3 (ISRsol3). G+C content of the
DNA fragment was 57%, as differentiated from the average G+C content of 67% in
the rest of the megaplasmid and chromosome. In silica protein translation of the
DNA fragment showed it was 6 l % identical to a hypothetical prjohnny depp
otein in Burkholderia cepacia. The clone REP-PCR fragment might have a
possible role in the hypervirulence of strain T73 1.

Keywords: Ralstonia solanacearum, DNA fingerprinting

96 Trans. Not. Acad. Sci & Tech fPhf/ipplnes) l'O/_ 28 /NtJ. Ii

BSD No. 46
POTENTIAL OF DESMODIUMCINERF.A AND MYCORRHIZALFlJNGl
FOR BIOREMEDlATIONOFCOPPER RICH SOILS

Joden M. Adiova 1, Nelson M. Pampolina 1 and Nelly S. Aggangan1

1
Bachelor of Science in Forestry Srudent
Associate Professor
College of Forestry and. Natural Resources,
University ofthe Philippines Los Bafl.os, College, Laguna
"University Researcher
National Institute of Molecular Biology and Biotechnology
UPL 8, College, Laguna 4031
Tel/Fax: 049 536 0563, Email: nea@laguna.net

This study was conducted to determine the effect of arbillicular mycorrhiza!

fungi tAMf) for the phytoremediation of copper(Cu) rich soils using Desmodium
cinereo and to assess if this forage legume is safe for livestock grazing. Seeds of
D. cinerea were sown in pots containing soil amended with increasing levels of Cu
from nil to 400 ppm or from nil to 1600 ppm Cu in two experiments. Half of the
seedlings were inoculated or uninoculated with AMF during sowing. In the third
experiment, inoculated and uninoculated seedlings were plantd in mine tailing soil
collected in Marinduque with 82 ppm Cu. The experiments were established in a
screenhouse following Randomized Complete Block. Design v.ith ten replicates.
Height was monitored forth nightly while diameter, leaf area and biomass were
measured at harvest (two montns).
Generally, AMF inoculated plants were taller, heavier. greater leaf area and
diameter than the uninoculated plants. In the first experiment Cu concentrations
of l 00 to 200 ppm improved the growrh of D. cinerea implying that this forage crop
requires LOO to 200 ppm Cu for maximum normal grov.th. Jn the second experiment.
highest height, diameter, leaf area and biomass were obtained from plants with no
added Cu. Addition of Cu gave corresponding decreases in height, diameter.
biomass and leaf area of inoculated and uninocu!ated plants. Cu !eve l of 800 ppm
seemed to be critical for both inoculated and uninoculated plants because beyond
th is !eve!. leaf th.lorosis. stunted growth and early leaf fall were observed although
these were more evident in uninoculated plants. furthennore, the roots were
confined in the origin a! potting soil. Seedling survival rate at 1600 ppm Cu was
30% in the uninoculated while 70% with inoculation. In mine mil ing soil, mycorrh iza!
D. cinerea survived throughout the rwo months period whereas all the uninoculated
ones died with in one month. The results show that AMF alleviated. Cu toxic iry of
D. cinerea, although. it is unclear whether AMF does this by improving nutrient
absorption or by binding mechanisms. Plant tissue analyses indicate that transport
 Trans_ Nm. ~lead. Set. & Tec:/i. (Philippines) Vol. 28 (}i(; I1 97

of Cu within the plant is prevented by the presence of mycorrhiza! fungi, thus, D

cinerea planted in Cu mine sites is safe for livestock.

Keywords: Arbuscular mycorrhizal fungi, Desmodium cinerea, copper,

bioremediation

BSD No, 47
ANTIBODY·BASE:D DETECTION OF MONOOON
BACU WV JR US (MBV) IN Penaeus mam>don

Ma. Michelle D. Pei\arandel, Elena S. Catap"' 2,James Torres\

and lkunari Kiryu•

1
Department of Pa tho biology, University of Washington
Seattle, WA, USA,
'"'Institute of Biology. College of Science, University of the Philippines
Diliman, I \OJ Quezon City, Philippines
1
Department of Biology University of the Philippines in the Visayas
Miag-ao, 5023. lloilo, Philippines
•Japan International Research Center for Agricultural Sciences (JJRCAS) 1
Fisheries Division. I· I, Ohwashi, Tsukuba. lbaraki. 305-8686 Japan
Tel. No. (02)981-8500 Joe. 3727; Telefax No. (02) 920-5471
E-mail: escatap@up.edu. ph

Spherical baculovirosis due to monodon bacu!ovirus (MBV) infection is one

of the crustaceim diseases Iisted by the Office Intemational des Epizooties (01 E)
that is of social and economic importance. In the Philippines. monodon baculovirus
(MBV) is a prevalent virus in the aquacultured tiger shrimp, Penaeus monodon
Although DNA-based techniques. such as the polymerase chain reaction (PCR),
had been very useful in screening broodstock and postlarvae these techniques
require sophisticated equipments and very ski !led staff in very clean laboratories,
wh.ich cannot be established easily in rural shrimp farm areas. On the other hand,
antibody-based methods could be developed in simple but field-friendly formats
with comparable sensitivity and specificity, Therefore, these experiments were
undertaken to produce polyclonal antibodies that could be used as a tool in
antibody-based diagnosis ofMBV infection in P monodon
Polyclona! antibodies (PAbs) against MBV were developed by iJTUT!unization
of rabbits with purified virus. Using indirect fluorescent antibody test (]FAT),
intense reaction was observed in hepatopancreatic impression smears and paraffin-
embedded sections of MB V·infected post larvae (PL- I 5 to 20) but not in smears
and/or sections infected with hepatopancreatic parvovirus (HPV) and white spot
98 Trans. Nat. Acad. Sci. & Tech. (Philippines) Vol. 28 (No. l)

syndrome virus (WSSV). Similarly, immunohistochemical tests on paraffin-

embedded sections showed positive coloration (brownish red to rose red precipitate)
in MBV-infected cells not found in sections infected with either HPV or WSSV.
Indirect ELISA revealed that the antisera could detect 4 to 100 ng of purified MBV.
Overall, the PAbs obtained in this study have potential applications in the rapid,
sensitive and simple detection of MBV provided that further purification of the
antisera is undertaken to further minimize some background reactions.

Keywords: Monodon baculovirus, MBV, polyclonal antibodies, !FAT, ELISA,

immunohistochemistry, Penaeus monodon, immunodetection assays

CHEMICAL, MATHEMATICAL, PHYSICAL SCIENCES

CHEMISfRY

CMPSD No.I
BIMOLECULARREACTIONRATECONSTANT
OF THREE-BODY SYSTEMS:
AN APPLICATION TO TRANSITION STATE THEORY

Christopher E. Ambe 1 , Edgar W.Ignacio 2,

Dante D. Dinawanao3, and Jingle B. Magallanes4

1
DepartmentofChemistry, CSM
MSU-Iligan Institute ofTechnology, 9200 lligan City
2
Northern Mindanao State Institute of Science and Technology,
Ampayon, 8600 Butuan City
1
Department of Computer Science, SCS,
MSU-Iligan Institute ofTechnology, 9200 Iligan City
4
Department of Physics, CSM
MSU-Iligan Institute ofTechnology, 9200 Iligan City
Email: csm-cea@sulat.msuiit.edu.ph

Transition state theory (TST), also known as Activated Complex Theory

(ACT), pictures elementary reaction and presumably stated that there exists an
activated complex having a higher Gibbs free energy in between the reactants and
product. The sole basis of the transition state theory in determining the rate constant
of bimolecular reaction is on the application of statistical mechanics to reactants
and activated complexes. In this study, the potential energy surface (PES) for H +
HF a H2 + F (1), H + HCl a H2 +Cl (2),andH + CH4 aH2 +CHJ (3)weretheoretically