phylogenetic analysis and prey identification of spiders...

Phylogenetic analysis and prey identification of spiders from wheat fields using CO1 as molecular marker

By Gulnaz Afzal

M. Phil. Zoology and Fisheries

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy In

Zoology and Fisheries

Department of Zoology and Fisheries FACULTY OF SCIENCES

UNIVERSITY OF AGRICULTURE FAISALABAD,

PAKISTAN 2013

Declaration

I hereby declare that the contents of the thesis, “Phylogenetic analysis and prey

identification of spiders from wheat fields using CO1 as molecular marker” are products of my own research and no part has been copied from any published

source (except the references, standard methods and protocols etc). I further

declare that this work has not been submitted for award of any other

diploma/degree. The University may take action if the information provided is

found inaccurate at any stage. (In case of any default the scholar will be proceeded

against as per HEC plagiarism policy).

Gulnaz Afzal

2002-ag-1113

The Controller of Examinations, University of Agriculture,

Faisalabad.

“We, the supervisory committee certify that the contents and form of this thesis submitted

by Miss Gulnaz Afzal, Reg. No. 2002-ag-1113 have been found satisfactory and recommend that

it be processed for evaluation by the external examiner(s) for award of the degree”

SUPERVISORY COMMITTEE

CHAIRPERSON : _______________________________

(Prof. Dr. Shakila Mushtaq)

MEMBER : _______________________________ (Prof. Dr. Shahnaz Akhtar Rana)

MEMBER : _______________________________

(Prof. Dr. Munir Ahmed Sheikh)

DEDICATION

To the grand pillars of my life: My parents:

Without you, my life would fall apart…

Abu Jan, you have given me so much, thanks for your faith in me and for teaching me that I should never surrender.

Ami Jan, you always told me to “reach for the stars.” I got my first one I think. Thanks for inspiring my love for transportation.

We made it…..

I

ACKNOWLEDGEMENTS

First of all I would like to bow my head before “ALMIGHTY ALLAH” the

compassionate and merciful, Who enabled me to contribute a drop of material to existing ocean

of scientific learning. I offer the humblest thanks from the core of my heart to the Holy Prophet Hazrat Muhammad (PBUH) the most perfect and exalted among human race who is ever lasting torch of guidance and source of knowledge for the humanity.

This dissertation would not have been possible without the guidance and the help of

several individuals who in one way or another contributed and extended their valuable assistance

in the preparation and completion of this study.

First and foremost, my gratitude to my supervisor Dr. Shakila Mushtaq, Professor, Department of Zoology and Fisheries, University of Agriculture, Faisalabad whose expertise,

understanding and patience, added considerably to my research experience. I appreciate her vast

knowledge and skill in many areas (e.g., vision, aging, ethics, interaction with participants) and

her assistance in writing reports (i.e., grant proposals, scholarship applications and this thesis),

which have on occasion made me "GREEN" with envy. Who until her day of retirement had kind

concern and consideration regarding my academics.

I am indebted to the other members of my committee Dr. Sahnaz Akhtar Rana Professor, Department of Zoology and Fisheries and Dr. Munir Ahmad Sheikh, Professor, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad for the

assistance they provided at all levels of the research project. They have been my inspiration as I

hurdle all the obstacles in the completion this research work.

It is difficult to overstate my gratitude to my technical advisor Dr. Amer Jamil,

Professor, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad for

his excellent guidance, caring, patience and providing me with an excellent atmosphere for doing

research. I doubt that I will ever be able to convey my appreciation fully, but I owe him my

eternal gratitude.

II

I am particularly obliged to Dr. Muhammad Javed, Chairman, Department of Zoology and Fisheries, University of Agriculture, Faisalabad who was willing to participate in my final

defense committee at the last moment. Indeed, his contribution of standards of academic and

research are immaculate.

I wish to thank Ghulam Mustafa (Junior Boss), who let me experience the research. His sincerity and encouragement I will never forget.

I am grateful to Dr. Aziz Mithani, Professor, SSE, Lahore University of Management and Sciences, for his expertise in Computational Biology and Bioinformatics. Despite the

distance, he has painstakingly e-mailed the information I needed.

I wish to thank Dr. Muhammad Mehmood_ul_Hasan, Associate Professor, Department of Zoology and Fisheries, University of Agriculture, Faisalabad for the assistance on how to use

the software needed for my research data analysis.

I am thankful to Abid bhai, lab attendant, Molecular Biology Lab., Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad who in one way or another

was always ready to provide his humble assistance especially when something was missing or

out of reach and if any system broke down during the course of usage.

I must also acknowledge my best friends Ahsan Rehman, Amber Irshad, Irum

Qadeer, Dr. Shazia Yasmeen, Dr. M. Samee Mubarik, Dr. Zubair Anjum, Dr. Muhammad Saeed, Dr. Shazia Perveen, Dr. Shamyla Nawazish, Dr. Shumaila Kiran and Dr. Shamyla Akhtar for their patience and steadfast encouragement to complete this study. They were always there cheering me up and stood by me through the good times and bad.

My extreme affiliation rests with my fellow students (Soma, Shaeen, Khan, Falak, Jabeen, Saba, Nazi, Maida, Samia, Ayan, Salman, Imran, Awais and Mustafa) for company, commiseration and countless lab/outside/coffee, tea, pizza, samosa, mango and tarbooz parties.

Thanks to all.

Last but not the least, about my family for telling me they were proud and for sharing my

excitement and somehow keeping their eyes from glazing over during long-winded scientific

rants.

Gulnaz Afzal

III

TABLE OF CONTENTS

No. Title Page No.

I

II Acknowledgements

Table of contents

I

III

III List of Tables VI

IV List of Figures VI I

V List of appendices III

V I Abstract IX

1 Introduction 1

2 Review of literature 5

2.1 Agriculture and biological diversity 5

2.2 Wheat agro-ecosystem and insects influx 5

2.3 Integrated Pest Management (IPM) 6

2.4 Spider’s role in IPM 7

2.5 Coexistence of species 9

2.6 Molecular identification of species 11

2.7 DNA barcoding 12

2.8 Predator-prey relationship 14

2.9 Molecular identification of prey in predators gut 14

3 Materials and methods 18

3.1 Study area 18

3.2 Collection of Spiders 18

3.3 Utilization curve 19

3.4 Estimation of niche overlap 19

IV

3.5 Estimation of niche breadth 19

3.6 Molecular studies 20

3.6.1 DNA extraction 20

3.6.1a Lysis buffer reagents 21

3.6.1b 5 % Sarcosyl solution 21

3.6.1c 10 M Ammonium acetate 21

3.6.2 Confirmation of isolated DNA 21

3.6.3 DNA quantification 22

3.6.4 Polymerase Chain Reaction (PCR) 22

3.6.5 Primers designing 22

3.6.6 DNA sequencing 24

3.6.7 Bioinformatic analysis 24

3.6.8 Deposits to GenBank 24

4 Results 25

4.1 Taxonomic and ecological studies Section I 25

4.1.1 Predators recorded from December through April 2008 25

4.1.2 Predators recorded from December through April 2009 25

4.1.3 Predator species dominance 26

4.1.4 Insect prey recorded from December through April 2008 28

4.1.5 Insect prey recorded from December through April 2009 29

4.1.6 Prey taxa of nine synoptic spider species 29

4.1.7 Predator prey records 39

4.1.8 Coexistence of common species 40

4.1.8.1 Utilization curves of nine synoptic spider predator species 40

4.1.8.2 Niche breadth 41

V

4.1.8.3 Diet overlaps 43

4.2 Molecular studies Section II 45

4.2.1 Isolation of genomic DNA 45

4.2.2 DNA quantification 46

4.2.3 DNA amplification 46

4.2.4 DNA sequencing and identification of species 49

4.2.5 Phylogenetic analysis 50

4.2.6 Neighbor Joining tree 51

4.2.7 GenBank sequences submission 53

4.2.2 Prey detection within predators 53

5 Discussion 55

5.1 Taxonomic and ecological studies Section I 55

5.2 Molecular studies Section II 60

6 Summary 63

7 Conclusions and Future Plans 65

7 References 66

8 Appendices 84

VI

LIST OF TABLES Table Title Page No.

4.1 Relative abundance of dominant predators caught during 2008 and

2009

28

4.2 Order wise prey consumed by Neoscona mukerji and its utilization curve 30

4.3 ………………………………...Argiope aemula..................................... 31

4.4 ........................................... Leucauge decorata ............................... 32

4.5 ........................................... Plexippus paykulli ................................ 33

4.6 ...........................................Cyclosa spirefera ................................. 34

4.7 ...........................................Oxyopes javanus................................... 35

4.8 ...........................................Hippasa olivacea.................................. 36

4.9 ...........................................Pardosa timida .................................... 37

4.10 ..........................................Tetragnatha javana............................... 38

4.11 Predator prey records, visual observation 2008 through 2009 39

4.12 Overlap values between nine pairs of synoptic spider species 44

4.13 Quantification of isolated DNA from nine synoptic spider species 46

4.14 Identified and maximum resembled spider species by nBLAST

analysis

50

VII

LIST OF FIGURES

Figure Title Page No. 4.1 Utilization curves of nine synoptic species computed from data in

Table 4.11

41

4.2 Niche breadth values of nine synoptic species 42

4.3 Agarose gel (1 %) for genomic DNA of spiders. (a): Lane 1 to 5

representing the N. mukerji, A. aemula, L. decorata, P. paykulli and P.

timida, respectively and (b): 1-4 representing the T. javana, H.

olivacea, O. javanus and C. spirefera, respectively. M: DNA ladder

(Fermentas, 1 kb)

45

4.4 PCR amplification of CO1 regions. Lane 1-3 showed PCR results of

N. mukerji, A. aemula and P. paykulli respectively. M: DNA ladder

(Fermentas, 1 kb)

47

4.5 PCR amplification of CO1 regions. Lane 4 and 5 showed PCR results

of L. decorata and C. spirefera. M: DNA ladder (Fermentas, 1 kb)

47


of O. javanus and T. javana. M: DNA ladder (Fermentas, 1 kb)

48


of P. timida and H. olivacea. M: DNA ladder (Fermentas, 1 kb)

48

4.8 Phylogenetic unrooted tree of nine spider species as inferred from

mitochondrial CO1 sequences analyzed by Neighbor Joining method,

Bootstrap values less than 50 % collapsed.

52

4.9 Lane 1-9 representing the agarose gel electrophoresis of PCR-

amplified DNA using insect specific CO1 primers. M: DNA ladder

(Fermentas, 100 bp)

54

VIII

LIST OF APPENDICES

Appendix Title Page No. 1 Reaction mixture setup for PCR to amplify CO1 gene sequences 84

2 Name and number of active spider predators belonging to different families, genera and species caught manually in different months from

wheat fields during 2008

85

3 Name and number of active spider predators belonging to different families, genera and species caught manually in different months from

wheat fields during 2009

88

4 Predators belonging to different families, genera and species caught manually during different months from wheat fields in 2008 and 2009.

90

5 Identification of prey belonging to different orders, families and

genera caught manually in different months from wheat fields

during 2008

96

6 Identification of prey belonging to different orders, families and

genera caught manually in different months from wheat fields

during 2009

98

7 Prey belonging to different orders, families and genera caught

manually during different months from wheat field in 2008 and

2009

100

8 Sequences alignment for nine spider species (PHYLIP interleaved

file format)

102

IX

ABSTRACT

Interspecific competition occurs among sympatric species when the availability of shared resources is reduced in the environment. Resource partioning (prey groups) among nine agrobiont spider species along their exact identification of prey and predators were verified in University of Agriculture, Faisalabad-Pakistan. This study based on predation evidences is highly supportive to compute coefficients of niche breadth and niche overlap. All overlap values were

1

CHAPTER 1 INTRODUCTION

Pakistan is an agriculture land, having diverse climatic and ecological

background. About 65.9 % populations depend directly on agriculture and play an

important role in growth, poverty alleviation and environmental protection (Bhutto and

Bazmi, 2007). Wheat (Triticum aestivum L.) is one third staple food of the world

population, currently the most grown crop among others i.e. rice, maize and potato

(Webb, 2000; Curtis, 2002). This crop is most vantage and pivotal for Pakistan. Major

wheat area in the country lies in Punjab followed by Sind. Surplus yield has resulted in

export to earn foreign exchange an 8.5 Mha of wheat is needed to feed hundrads million

people every year (Anwar et al., 2009). Wheat is encountered with many potential

problems as well; the most intractable one is reduction in wheat yield by insects. Insect

pests along their predators are evident in wheat fields of Pakistan, few species may

become pests and they usually cause damage above threshold level. However, various

major and minor insect pests of wheat may become abundant enough to damage the

wheat crop significantly such as aphids, cereal leaf beetle, hessian fly, armyworm,

grasshoppers and chinch bugs (Salim et al., 2003; Ramzan et al., 2007). Control of such

invasive insects is always being monitored by chemical and biological bases where

indiscriminate use of such chemicals produces toxic residues with severe and costly side

effects on human health and environment. In addition, insects become resistant to

insecticides, limiting their long-term efficacy, decimating populations of non-target

beneficial organisms. Agricultural productivity could be enhanced by controlling insect

pests (Benton et al., 2002).

Mobilization of biological control is a fundamental campaign to suppress the pest

population within an effective Integrated Pest Management (IPM). In biological control,

natural enemies are being brought into play against a pest population to reduce its density

and damage to a level lower than that would occur in their nonattendance. On account of

its effectiveness IPM is increasingly becoming very popular, approximately 75 %

generalist predators whether single or bulk of it is sufficent to reduce pest populations

significantly in agro-ecosystems (Symondson et al., 2002).

2

To design an effective IPM, identification of predator-prey species and their best

fitness in fields is important. Spiders (“Phylum Arthropoda, Sub-Phylum Chelicerata,

Class Arachnida and Order Araneae”) are a versatile group, found universally from

seashore to mountains and from timberland to agri-ecosystems (Foelix, 1996). Mostly fall

in the group of predaceous organisms in animal kingdom; these arachnids have been

demonstrated as an effective component of IPM programs, which combine the natural

insect predators with judicious use of insecticides. Spiders tend to concentrate on insect

prey and to a lesser degree on other spiders (Wise, 1993) which make them ideal as bio-

control agents, along with other generalist predators, mainly accounting for supporting

biocoenotic constancy.

While generalizations may not apply to all species within a taxon, most of the

spiders are predominantly generalist predators that may have evolved their particular

niche exploitation patterns under different ecological circumstances by exploiting the

same class of resources. In view of the fact that potential prey in agroecosystems may

vary with microhabitat, season, time of day and foraging strategy of spiders. Spiders

possibly will constitute more than one assemblage guild, which represents a strong mirror

taxonomic relationships. So, that assemblage of generalist predators can impact pest

populations and reduce crop damage (Riechert and Bishop, 1990; Uetz et al., 1999).

Accordingly, it is important to recognize how much predator and prey species are well

matched in the agro-ecosystems and employ these predators as pest control agents.

Spiders prey utilization is naturally very difficult due to some of its ubiquitous

attributes, very small, having extra oral digestion, sucking mouthparts and amorphous gut

contents. Spider’s digestive enzymes partially liquefies their prey externally and suck up

liquid digestion product (usually less than 1 micron) containing a few morphological

remnants, as molecular and biochemical remains present in sucked liquid. Molecular clue

is the presence of DNA of recently consumed prey by spiders (Agusti et al., 2003).

Similarly, in this way amplification of nearly 486 bp sequence of a mitochondrial CO1

(Cytochrome C Oxidase 1) gene with correct primers is used for identification of prey

(Dunshea, 2009).

3

Identification of species helps to make strong background about how to

understand the diversity, phylogenetic patterns and evolutionary processes (speciation

and extinction) among and between the species. Only accurate identification allows for

comparison and expansion of earlier experiments (Cohn, 1990). Exact identification

based on morphological characteristic always remained a problematic issue. This

complexity accounts for the major obstructions to identify the species. First, the keys

followed, mostly rest on the inspection of adults; secondly, the common existence of

marked sexual dimorphism produced severe problems of synonymy, third, identification

is significantly limited by phenotypic plasticity, genetic variability and inability to detect

the cryptic species and at the end taxonomic keys demanding an extraordinary level of

proficiency (Huber and Gonzalez, 2001; Jocque, 2002).

Because of all above-mentioned problems it was critical to develop an adequate

system of identification which may help to solve these problems and broadly acceptable.

Bio-molecular studies offer an accurate solution to these problems (Tautz et al., 2002;

Hebert et al., 2003; Blaxter and Floyd, 2003). Molecular taxonomy is serving as a slogan

for the existing 21st century’s systematics. For this purpose DNA barcoding is currently

proved as an indispensable tool in the box of biotechnology for diet analysis, delineating

and for allocation of unidentified specimens to specific edging, to explore the criptic

species and to augment the prospects to encounter new species using a threshold of

sequence deviation (Moritz and Cicero, 2004; Savolainen et al., 2005; Monaghan et al.,

2005). In DNA barcoding, usually a sequence at 5ʹ end of the mitochondrial CO1 is used to categorize the species (Berret and Hebert, 2005; Hajibabaei et al., 2006). Predation

always remains one of the most difficult ecological processes to study because evidences

can be gathered only by direct observations in fields that takes too much time and

attention to locate and sort out the spider along its prey in their mouth but more recently,

biomolecular studies has become a least disruptive and most efficient approach in context

of identifying direct feeding of farmed pest by predator (Caterino et al., 2000; Harper et

al. 2005). In such context CO1 befalls in manifold copies per cell, which raises the

probability of effective amplification of gut contents of species. DNA based identification

is more rapid and unambiguous over traditional morphological means; one can use

4

material from any stage of the life cycle, reducing the need of time cost and risky practice

of rearing insects (Leigh et al., 2008). PCR amplification is simpler and more versatile to

make the possible use of any insect material such as fragmented and preserved DNA

remains (Marrelli et al., 2005; Li et al., 2010; Guerao et al., 2011).

It can be prepared highly operative by selecting primers that amplify short,

multiple-copy fragments through DNA barcoding. Mitochondrial genes have been

revealed effortlessly demonstrable targets concerning to check the systematics and unlike

level of preservation of different genes and part of genes (Chen et al., 2000; Agusti and

Symondson, 2001).

Finally, system of molecular taxonomy is an advanced technique over pre-

existing morphological framework and it is extremely helpful to narrow the gaps in the

field of molecular taxonomy, which in turn would broaden our knowledge about the

spiders and makes possible exact identification through DNA analysis. Only a handful of

papers from Southeast Asia have been reported till now which conferred information

regarding molecular phylogeny of spiders and their prey through DNA barcoding (Fang

et al., 2000; Robinson et al., 2009; Su et al., 2011; Krishnamurthy and Francis, 2012).

However, from Pakistan to date, such kind of bio-molecular taxonomy is not reported.

Therefore, it was dire need to plan such type of studies in Pakistan. On the bases of

molecular approach that utilized CO1, present research work was proposed to investigate

the faunistic quantification of the araneids in wheat fields in Pakistan, along with prey

these spiders were found to consume. This attempt will meagerly provide a starting point

to re-evaluate the Pakistani spider’s fauna on bio-molecular bases. Objectives of the

study were as follow:

Objectives Record, morphological identification of predator/prey species occurring in wheat.

Establish predator’s phylogeny using CO1 molecular marker

Recognition and identification of prey within gut contents through PCR using

insect specific primers.

5

CHAPTER 2 REVIEW OF LITERATURE 2.1 Agriculture and biological diversity

Pakistan lies between 24.0◦ and 37.0◦ North latitude and 60.0◦ and 75.0◦ East

latitude, approximately covering 8,27,048 Km2. As it lies in Southeast Asia, the climate is

diversified, augmenting four seasons which exhibits some variety among them. It has

remarkable world’s ecological regions along their great geological history, very vast

biodiversity which is a blend of elements from Palaeartic, Oriental and Ethopion regions

resulting into eighteen distinct types of natural habitats (CIA, 2010).

Biological diversity included all components of food, different plants, animals,

microorganisms and ecosystem level which are essential to tolerate important roles in the

agro-ecosystems (Altieri, 1999). The agro-ecosystems which have differences in

management, age, structure and diversity also have differences in type and abundance of

biodiversity. In addition to food production, fiber, fuel and income, biodiversity is

performing a variety of ecosystem services including nutrient recycling, management of

local micro climate and detoxification of hazardous chemicals. The tenacity of these

biological processes depends upon stability of biological diversity within ecosystem

(Tilman, 2000). By conserving the biological diversity a sustainable agriculture could be

maintained with long term yield production (Van der Putten et al., 2000). Agriculture is

backbone of the Pakistan and has become its largest sector of national economy. It is

strongly linked with food security, poverty alleviation, rural development and best

employment opportunities. Presently, it pays 16.2 million workers, who represent 47.5%

of total labor force. It is also an important source to bring the lucrative foreign exchange

by exporting the agricultural commodities and products (Sheikh et al., 2005; Sheikh,

2008).

2.2 Wheat agro-ecosystem and insects influx Most of the area of Punjab Pakistan is under the cultivation of different types of

crops such as maize, potato and rice. Among all other agricultural crops, wheat (Triticum

aestivum L.) is one third staple food of the world population (Webb, 2000; Curtis, 2002;

Anwar et al., 2009) second in significance after livestock and just in advance of

horticulture. It is an important service in the nutrition of those who are underneath the

6

poverty line and more usually it implies a thoughtful establishment in expressions of its

influence to national food security. Wheat is a key element enabling the emergence of

city-based societies from start of the civilization. It is first crop that could be simply

cultivated on a big level, having an extra gain and ultimately would offer very long-term

storage. It is a most pivotal for Pakistan, very fast in growth with 14% of incremental

development in agricultural GDP. Wheat characterizes an outstanding contender for

direct applied investigations, extensions that would be highly relative to expenditure in

reappearance (World Bank, 2007).

Under cultivation areas provide habitat for different kinds of invertebrates that

constitute a major part of an agro-ecosystem. Even though, they are useful bio-indicators

of agro-ecology nevertheless cause a lot of potential problems as well. Insects are

responsible to damage the fields in direct or indirect means, which results in reduced total

wheat yield in country. The bulky inhabitants and great diversity of insects are associated to their trivial size, high rates of imitation and plenty of appropriate food supplies. Insects

proliferate in the tropics, both in numbers of different varieties and individuals. There are

hundreds of such type of pests, related to orthopterans, homopterans, heteropterans,

coleopterans, lepidopterans and dipterans. Insect pests along their predators are evident in

wheat fields of Pakistan. Only a few species may become pests and they usually cause

damage above threshold level. However, various major and minor insect pests of wheat

may become abundant enough to damage the wheat crop, among them spiders are

dominant (Salim et al., 2003; Ramzan et al., 2007).

2.3 Integrated Pest Management (IPM) In recent decades, agricultural practices towards expansion and intensification

includes treatment with large quantities of chemicals viz fertilizers, herbicides, pesticides

and fungicides etc. These all are the major threats to biodiversity (Benton et al., 2002).

This critical loss of biodiversity may lead to changes in ecosystem functioning and

resilience of agricultural systems. Such chemical control approaches defend crops from

being spoiled by pests, they utterly diminish biodiversity. According to FAO

assessments, in the next decade, about 90% of standing genetic biodiversity within chief

crops is at stake (Mozumber and Robert, 2006). Moreover, such agro-chemicals produce

7

toxic residues with severe and costly side effects on human health regarding too many

neurological disorders and few types of cancers (Morgan, 2005). Agricultural

productivity could be enhanced by controlling insect pests. A pest problem intensifies

when natural predators are removed by pesticides. Pesticides can damage soil, reduce

food availability and habitats of arthropods that are source of food for others. Specific

doses have been recorded as source of organ deformities in vertebrates in different states

of Florida (Pretty, 1998; Bourne, 1999).

Mobilization of biological control is a fundamental campaign to suppress the pest

population within an effective Integrated Pest Management (IPM). It is a flexible

approach to crop protection; an approach that makes best use of all available technologies

to manage pest problems effectively, securely and sustainably. IPM subsequently

integrate all appropriate measures that disappoint the growth of pest populations and keep

pesticides and other involvements to levels that are economically acceptable and lessen

risks to human health and environment. IPM is being increasingly accepted due to its

effectiveness, approximately 75 % generalist predators whether single species or bulk can

reduce pests in agro-ecosystems (Symondson et al., 2002; Maloney, 2003).

2.4 Spider’s role in IPM To design an effective IPM, identification of predator and prey species and their

best fitness in fields is important. For engaging spiders in the IPM, it is nesessary to

recognize more about their biology, classification and variety in local agro-ecosystems.

Morover, awareness of key species of a community about their size, sex ratio and

abundance may deliver valuable ecological facts that could be highly promoted in

considering the predatory potentials. Spiders fall under natural control factors. They form

a substantial constituent of food web in both the natural and man-made agro-ecosystems.

Approximately 37,000 known spider species are currently found in the world and these

are acknowledged as a fraction of overall diversity (Adis and Harvey, 2000). According

to another estimate by Platnick (2012) a total of 43,244 species have been counted uptill

now. Spiders have been reported feeding on a wide variety of insect pests by adopting

different means including feeding on arthropod eggs, dead remains, plant pollen and even

artificial diets (Nyffeler et al., 1990a). Predominantly they are polypagous in nature and

8

mostly feed on small sized prey relative to their own body size and highly selective in

nature to extract the essential amino acids in their diet (Nyffeler and Benz, 1981). They

have great biodiversity and found remarkably dominating among other memebers of the

community (Butt and Beg, 2000, 2001). They may be distinguished into two groups due

to their foraging practices: (1) Web spiders foraging with grasping web and (2) hunters or

wanderers missing the help of web. They form one of the most universal groups of

predaceous creatures in the animal kingdom. They feed almost entirely on insects and

their larvae by killing them through disturbance. Spiders can exterminate more pests than

commercial insecticides that may lead to a 60% reduction in chemical use (Marc et al.,

1999).

Spiders are responsible to play their role in two ways in every ecosystem. First,

they perform their predatory pressure on entomocoenosis and on some particular insects.

Under crop conditions, they are best rivals of aphids, mites and lepidopteran species.

Spiders along with other general predators are much responsible to withstand stability in

ecosystems and have been used as the natural predators of insects in orchards and in rice

fields of Japan, Republic of China and Republic of Czech (Pekar and Cocourek, 2004).

Jones (1981) stated that straw bundles could be used to provide shelter for spiders and

transported from crop to crop as needed to implement control. To record spiders

biodiversity a survey of foliage and ground spider fauna of Punjab Pakistan was made

from 1996 through 1998 by Parveen (2006) spiders were collected from 21 districts

(Faisalabad, T.T. Singh, Jhang, Sheikhupura, Lahore, Gujranwala, Sialkot, Jehlum,

Rawalpindi, Chakwal, Sargodha, Okara, Sahiwal, Khanewal, Multan, Muzaffar Garh,

Bahawalnagar, D.G. Khan, Vehari, Leiah and Rahim Yar Khan) of Punjab and also from

one location in Federal Territory, Islamabad. A total of 14,743 spiders were captured,

belonged to 21 families, 58 genera and 157 species. Out of these, 80 species have never

been logged from these sites and 32 were cognized as novel to the science. A similar

study was also conducted in 1996 to 1998 and 2000 by Mukhtar and Mushtaq (2005). At

this time genus Clubiona was the major focuse, a record of five species belonged to 99,

62 and 79 females, males and immatures respectively were documented. There was also

an addition of two new species in the literature of Araneae systematics; Clubiona

9

kasurensis Mukhtar and Mushtaq 2005 was new to science and Clubiona filicata O. P.

Cambridge 1874 was reported first time in Pakistan.

In addition, a very little spider’s fauna of Pakistan has been reported till now

regarding to their taxonomy, ecology and economic importance (Qadir, 1997; Mushtaq

and Qadir, 1999; Butt and Beg, 2001; Ghafoor and Beg, 2002; Razzaq, 2002; Tahir and

Butt, 2009; Perveen et al., 2012). It is supposed that there are a lot of species which have

not yet been discovered. Practical applications of spiders in Pakistan can save billions of

foreign exchange, help to reduce severe environmental fluctuations and lethal effects on

human beings caused by insecticides.

2.5 Coexistence of species Due to their high colonization and predation rate, spiders have snatched great

interests of scientists especially concerned with issues of natural biological control,

resource partitioning, interspecific competitions to coexist in the same environment

(Sterling et al., 1989). Agro-ecosystems are variable environments with wide niche

dimensions that reduce the niche compition among species and allow them to coexist.

Niche divergences are the result of directional selection, if resources are abound in

supply, different species can share them without detriment to one another and niche

overlap may be high with reduced competition (Molles, 2007). Partitioning of resources

also occurs between sexes of the same species, different body sizes within species, as

well as across species (Belk et al., 1988). Competition is the foremost basis that figures

the structure of a community. Because of different patterns in natural communities such

as different resource utilization, morphological deviations, variable life histories,

isolation in activity times and allopatric dispersals can be illuminated within the context

of competition (Toft and Schooner, 1983).

Resource expenditure in cluster living of spider’s community, species are of

similar body size, with behavior ranging from near solitary to fully social, always

cohabit. Captured insect size by each species reflects their web building and colonization.

Species with larger colonies captured larger insects than less social species. It also

depends on the variable extent of nest mate’s cooperation in prey capturing. If species are

10

more gathered, they avoid extensive dietary overlap then there would be more over

dispersed resource utilization expected by chance (Guevara at al., 2011).

The most dominant objective of ecology is to apprehend forces that sustain

species diversity within communities. Struggle for food has long been deliberated as

bedrock of community ecology. Cohabiting of species at same area always requires some

sort of resource sharing between them to prevent interspecific competitions (Davies et al.,

2007). Based on the exclusion principle (Complete competitors cannot coexist), the most

successful species of spiders should drive others to extinction. Because there is more than

one species of spiders living sympatrically, they partition the resources in some way that

decreases niche overlap and permits for coexistence (Perkins, 2009).

Ecological indices of M. menardi Latreille 1804 and M. merianae Scopoli 1763

(Tetragnathidae) were estimated by Novak et al. (2010). They reported that both species

coexist in the entrance section of the studied cave and can part its relative by abundant

resources. One of them is synoptic and sympatric in comparison of other. Significantly,

with typical r-strategy epigean dynamics in M. merianae was high and a steady low in M.

menardi within the cave. In addition, their spatial niches are very similar but trophic

niche of M. menardi is broader due to its heavy prey mass, wider or extended list of prey

that species was suggested as optimally best adapted to live in the hypogean ecotone

zone.

Moreover, resource utilization can befall in different methods: species might

fluctuate in where they exercise and retort to a limiting factor, different species may be

narrowed by the same resources, but fluctuate in time when they deed resources on their

demand so, co-occurring species may specify in different resources. Such types of

partitioning would be the outcome of selection for ecological character divergence among

sympatric populations (Chesson, 2000; Dayan and Simberloff, 2005; Davies et al., 2007).

Differences in size may also be attributable to character shift that may have arisen in

morphology before the species would become sympatric. This preliminary stage is likely

to be a chief funder to stable cohabitation of potential competitors (Guilleman et al.,

2002; York and Papes, 2007). The niche axes of two endemic sympatric desert species,

Syspira tigrina Simon 1885 and Syspira longipes Simon 1885 (Araneae: Miturgidae) in

11

the State of Baja California Sur, were evidenced that coexistence is bring up by variances

in choice of microhabitat, temporal activity, occupation of space or size (Nieto-

Castan˜eda and Jime´nez-Jime´nez, 2009).

A study was conducted to investigate the coexistence of two (hunting and orb

web) spider guilds in same area that never affect being changed in their microhabitat,

prey niche dimensions, and separation of guild members in time. Their resource

utilization overlap is differed with their abundance, reproductive period and prey size. It

is concluded that their conservation in rice fields would be meaningful to enhance the

biological control potential (Tahir and Butt, 2008; Tahir et al., 2009; Butt and Tahir,

2010).

2.6 Molecular identification of species Exact identification based on morphological characteristic always remained a

problematic issue. It has several limitations to identify the species including, phenotypic

plasticity of traits that lead to misidentification of species (Knowlton, 1993; Jerman and

Elliott, 2000), morphological keys mostly rely on particular life stages or gender based

(Henning, 1966). Thus, high levels of expertise are required to correctly identify the

species. Bio-molecular studies offer an accurate solution to all these mentioned problems

(Tautz et al., 2002, 2003).

Molecular identification is promptly becoming the slowgan of modern 21st

century’s systematics. It has a major advantage over conventional techniques of

microscopic examination that very small amount of material (hair, tissue, blood droplet

and a rasping of skin) is required to identify the species as compared to earlier techniques

Simon et al., 1994). Thus, it is much easier now to identify and delineate a single species,

a group of population and even an individual (Iverson et al., 2004; Green and Minz,

2005; Dunshea et al., 2008). Essentially, two types of molecules (protein and DNA) are

being used for molecular identification in ecology. Applications involving protein are

rather tough and expensive than those with DNA due to presence of less polymorphism.

Furthermore, protein analysis usually entails comparatively large quantities of sample

and remarkably encounterd less availability of different factors (space and time) (von

Wintzingerode, 2000). Several applications of this technology are being used now a day.

12

The areas of medical sciences and human forensic information have also been used as

convincing sign for diagnose of diseases, paternity, homicide and sexual assaults that has

become a marked request in commercial tests.

Molecular studies may be constructed on one or numerous mitochondrial and

nuclear regions. DNA taxonomy is a branch of phylogenetics, in which the evolutionary

associations between taxa are examined (Sunnucks et al., 2000). For monophyly with an

ancestral character reconstruction, mitochondrial 12s, 16s and CO1 DNA sequences are

proved accurate markers (Hannah et al., 2007). DNA built method is proficient to

accelerate the universal characteristics of a large number of insect species and their

developmental stages too. There is always an uncertainty to identify and delineate the

species due to genetic differences within them. This ambiguity can be triumph over by

establishing groups on DNA bases and adult-larvae associations. Sequence dissimilarity

in mitochondarial and nuclear DNAs is greatly prearranged and consequently, all

individuals (larval or adult) could be gamely coupled with a particular DNA group. After

establishing a particular group, morphological characters in larvae and adults would be

much easier to diagnose individually (Ahrens et al., 2007).

2.7 DNA barcoding The word DNA barcoding is new in the literature (Floyed et al., 2002). Use of a

standardized DNA region has recognized as a tag for swift and exact species

identification (Hebert and Gregory, 2005). DNA barcoding , a version of the DNA

taxonomy paradigm to arachnids and various other animal species, show that it is

possible to identify members of all existing animal fauna by using a short fragment of the

mitochondrial gene coding for cytochrome oxidase 1 (Blaxter, 2003; Hogg and Hebert,

2004). CO1 is selected for barcoding due to some upper hand attributes compared to

other mitochondrial protein coding genes; insertions are rare, very permissible for

sequencing of the animal phyla and having a fast rate of nucleotide substitution which

helps to make discriminations among cryptic species and phylogeographic structures

within a species as well (Pires and Merinoni, 2010).

In this background, it is essential to differentiate the DNA barcoding and

taxonomy. DNA barcoding resolves the identification of pre-defined species only while

13

DNA taxonomy addresses the issue of limitation and explanation of species by means of

evolutionary species concepts (Vogler and Monaghan, 2007). Most obvious application

of a molecular tactic has been proposed that, CO1 sequences are used in Neighbor joining

trees to barcode taxa. This approach is more advanced over traditional taxonomy systems

because morphological analyses are less or incomplete (Kress et al., 2005). The

taxonomy of genus Latrodectus Walckenaer 1805 revealed the first phylogenetic

hypothesis from DNA sequences of the mitCO1. It is evident that the recovery of L.

geometricus Koch 1841 and L. mactans Fabricius 1775 clades is reliable with former

descriptions of species within the genus created on female genitalic morphology (Jessica

et al., 2004).

It is evident that CO1 and 16s ribosomal genes are maternally inherited by all

animals and plants. In such context the sequenced data from the mitochondrial genome

has been used to assess monophyly of species with special reference to the status of the

putative hybrid species (Nadia et al., 2005). Single strand conformational analysis of

mitochondrial CO1 is also used to characterize the haplotypes of wolf spiders at different

locations. Genetic diversity is high as compared to nucleotide diversity, as all the

haplotypes are closely linked with each other, the inter population comparisons show

significant nonhomogeneity of haplotype frequencies that may have antedated habitat

fragmentation with no correlation geographically (Colgan et al., 2002).

Documented information on numerical morphological data composed with

phylogenetic relationships, re-evaluates the limitations of genus Havaika Proszynski

2001 taxonomy. The inferred phylogeny is based on DNA sequences, both mitochondrial

(CO1, NAD1, 16s and tRNA leu) and nuclear (internal transcribed spacer 2) genes. The

suggested information about Havaika may be the consequence of sovereign

colonizations. Furthermore, it provides a slight provision for standard progression rule in

Hawaiian Islands (Miquel and Gillespie, 2006).

Information regarding the resolution concerns of species delineation and

phylogeny of spider mites in the family Tetranychidae, central part of CO1 region has

often been used to explore the intra and inter-specific variations. This extensive database

of sequence information is assembled in a single alignment and accomplished in an

14

overall phylogenetic analysis (Ros and Breeuwer, 2007). Finally, DNA barcoding has an

extra ordinary prospective for immediate multiple-species documentation from a single

environmental sample, for biodiversity assessment and for predation inquiry from feaces

(Jarman et al., 2002).

2.8 Predator–prey relationship Insect pests control always remained a problematic issue over the last few

decades. Predator prey relationship is a widespread and complex food web issue of

agricultural crops. Predatory behavior of spiders is considered more significant while

playing an important role in reducing crop damage and insect pest’s populations (Stuart

and Greenstone, 1990; Greenstone, 1999). They can control caterpillar in cotton,

Colorado beetle, pests in rice and hoppers (Clark et al., 1994; Carter and Rypstra, 1995;

Nyffeler and Sunderland, 2003; Nyffeler, 2009). Use of a single predator to control more

than a few other species constructs a simple food web as compare to several prey against

numerous predator species. Sharing of two prey by a predator results in depleting the

density of one prey by increasing the density of other (Moris et al., 2004). Thus, an

indirect effect such as “apparent mutualism” is created if one species is in abundance and

available to predator with a decrease predation rate upon others, affects each other’s

density (Van Rijin et al., 2002).

2.9 Molecular identification of prey in predators gut In molecular identification, one of the most inflexible applied problems in ecology

remarkably is the study of prey-predator interactions. Such trophic link is difficult to

study directly in field conditions. Identifying prey and its range can be a frightening task

particularly with small organisms. Mostly examination of gut contents has to be done by

manual methods. Molecular methods offer a useful substitute and have newly been

employed to identify multiple possible prey items instantaneously. It is also essential to

have information about population dynamics, breeding biology and host switching of

predators and parasitoids for the control and minimum non-targeted effects on the

ambient environment and also on other populations residing in close associations

(Macdonald et al., 2004; Bigler et al., 2005). A range of different molecular techniques

and applications have been used. Which include enzyme electrophoresis, immunological

15

assays by polyclonal and monoclonal antibodies to discover protein epitopes. All these

methods are most effective, highly sensitive, expensive and multifaceted (Greenstone,

2006). Recently, PCR-based approaches for detection of prey in predator’s diet are

proven to be highly effective for the study of predator-prey relationship and are likely to

rapidly displace all others (Symondson, 2002; Harper et al., 2005).

Hence, DNA markers are certainly beneficial to detect prey presence in the

predator’s gut. A unique fragment of Greenhouse Whitefly (Trialeurodes vaporariorum

Westwood 1856) was found absent in allied prey species and predator was augmented by

Random Amplified Polymorphic DNA (RAPD) analysis. The Sequence Characterized

Amplified Regions (SCAR) marker was developed after cloning. In gut assays the CO1

has higher detection efficiency than other markers (Agusti et al., 2000; Lang et al., 2004).

It has been proposed by Harper et al. (2005) that more than ten potential prey of the

Pterostichus melanarius Illiger 1798 could be ascertained through molecular markers.

According to another study primers (taxon-specific) were been used to amplify short COI

genes from aphids and beetles, in multiplex PCRs. Wolf spiders (Araneae: Lycosidae) are

important predator of insect pests in fields. Their identification is tough especially in the

immature stages. By multiplex PCR, variations in the CO1 gene sequences among spiders

are diagnostic with respect to species identification (Hosseini et al., 2007). Similarly,

primers target both predator and prey species and help to screen out 100% detection of

recently consumed prey as compared to singleplex assays because it considerably reduces

the time and cost (Juen and Traugott, 2006).

DNA barcoding makes it promising to launch the diet of an individual from its

feces or stomach remanants. This is helpful when morphological standards are not

sufficient to identifiy food items, such as in fluid suckers like spiders (Caterino et al.,

2000). PCR-based applications have substantiated to be highly operative in recent

laboratory practices (Symondson, 2002 and Jarman et al., 2004). Multiple copy genes,

whether nuclear or mitochondrial, considerably surge the possibility and interval of prey

discovery within predators. Particularly, mitochondrial genes are more reliable for such

kind of work because much is well-known about levels of preservation of different genes,

being extensively used in insect phylogenetics (Chen et al., 2000).

16

A comparison of barcode fragments of cytochrome oxidase 1 with morpho-

ecological traits among North America tiger moths genus Grammia were illustrated that

such species sharing haplotypes are often not closely related but shows a high mtDNA

divergences within species. It is also inferred the value of mitDNA in detecting cryptic

hybridization. Hence, molecular evolution of Grammia interprets that interspecific gene

exchange arises infrequently and is constrained to newly diverged species (Schmidt and

Sperling, 2008).

Sequence divergence at the CO1 barcode region has also been shown to be

effective for discriminating European species of crab spider genus Maja at all stages of

development. Four species M. bracydactyla Balss 1922, M. squinado Herbst 1788, M.

goltziana d’Oliveira 1888 and M. crispate Packard 1864 were discriminated by applying

the mixture of restriction endonuclease enzyme HpyCH4V and ASE I. A novel

morphometric index and PCR-RFLP analysis of adult M. brachydactyla and M. squinado

were used to distinguish both species by finding the relationship between carapace

measurement and the distance between the tips of antorbital spines (Guerao et al., 2011).

Harwood et al. (2004) reported, arthropods predation on populations of prey in

fields, obtained by using PCR primers amplify DNA fragments from 211 to 276 bp in

length, was detected Collembola as an alternative prey within spider predators. It is

demonstrated that all the three Collembola (Isotoma anglicana Lubbock 1862,

Lepidocyrtus cyaneus Tullberg 1871 and Entomobrya multifasciata Tullberg 1871)

species were also being exercised prey choice by spiders (Agusti et al., 2003). Another

molecular approach regarding the selection of prey by Linyphiid spiders, the rate of

predation on aphids by Linyphiinae is correlated with its mass, not obtainability of other

prey. Similarly, predation of Erigoninae on aphids is expressively exaggerated by

Collembola mass (Harwood et al., 2004).

Moreover, the challenges of confusing primary and secondary predation is

particulary acute in fields that can also be monitored by CO1 amplification. A profound

PCR amplification may detect the prey used up by the predator, somewhat being openly

consumed by the predator itself. According to this background, the ground beetle

Pterostichus melanarius Illiger 1798 was fed with spiders that had previously eaten

17

aphids. Specific PCR amplification of 110 and 245 bp fragments of aphid COI DNA

showed that secondary predation were eagerly evident for up to eight hours after beetles

fed on spiders that had formerly consumed aphids (Sheppard and Harwood, 2005). This

credit goes to spider with reduced metabolic rates between feeds and prey DNA remained

detectable in them much longer than it did in beetle guts. Many other predation studies

may be less susceptible to the detection of secondary predation that may cause error in

food chain determination. Conclusively, molecular pinpointing gears have been

instrumented in the analysis of predator gut remanats, chiefly in the identification of prey

substances, assessment of predator favorites and regularity in predation. In addition, host-

parasitoid relations, inhabitant’s dynamics, intraguild predation and trophic

collaborations can be investigated by universal markers (Zhu et al., 2000; Pons, 2006;

Gariepy et al., 2007).

Indeed, agro-ecosystems would not properly function in absence of spiders. It is

found that spider’s biodiversity is an important factor in pest management strategies

rather than applying agrochemicals (Manoley et al., 2003). Pakistan is facing a

detrimental loss of biodiversity which ultimately affects all trophic levels of food web in

agro-ecosystems thus indirectly posing a stress on human life (Benton et al., 2002). For

implementing sustainable agricultural strategies, assessment of best fitted prey and

predators with their exact identification in agro-ecosystems are highly incredible. The

literature on molecular taxonomy of spiders in wheat fields of Pakistan is deficient and

also the role of spider’s predation specialization is lacking in making the system more

stable. With this background information the present study was planned to identify the

spider’s fauna on both morphological and molecular bases with special emphasis to

explore the literature gap in spider systematics and predator-prey potentials in wheat

fields of University of Agriculture, Faisalabad Pakistan.

18

CHAPTER 3 MATERIALS AND METHODS 3.1 Study area

Winter wheat fields were surveyed, during 2008 and 2009 December through

April, for araneid fauna at University of Agriculture Faisalabad, Pakistan. Total area of

research fields was 4060 m2 that was remained untreated with any sort of insecticides or

herbicides till the whole experimentation. All fields were harvested in May during both

years. The average temperature and relative humidity ranged 17.0-32.5, 55.5-33.6 (2008)

and 11.5-31.8, 68.9-41.7 (2009) respectively. Temperature and relative humidity data

were obtained from Agricultural Meteorology Cell, Department of Crop Physiology,

University of Agriculture, Faisalabad Pakistan.

3.2 Collection of spiders To inspect the active density of spider pitfall traps and a suction device were

applied throughout the growth period of wheat. For everyweek, twenty pitfall traps were

set and operated during December to April under 24 h observations to achieve the ground

spiders. Each trap was 14 cm long glass jar with 7 cm wide rounded mouth, buried in the

ground so that the upper rim was at level of the soil surface. Such plotted jars were filledwith a

mixture of 150 ml of 70% ethyl alcohol and a small quantity of kerosene oil. Both

materials were used for the sack of preservation and protection of captured contents. The

traps were found operational at daily checking and replaced with fresh ones after 48 h.

After the completion of another 72 hours all the traps were taken out.

Foliage spiders were collected using a suction device (Siemens VK 20C01) from

December to April. Wheat plants were randomly selected and vacuumed for 1 min

thoroughly. To obtain the predators with their insect prey (Prey in spider’s mouth) on

daily basis, sampling was done in morning and evening only for two hours. Observed

predation events were recorded and all captured predators along prey were stored and

brought to the Araneae laboratory, Department of Zoology and Fisheries. All captured

individuals were washed with xylene and preserved in 95% ethanol containing a little

quantity of glycerin. Collected spiders were identified based on traditional morphometric

characteristics. Insect prey was identified into order, family and generic level only while

predators were identified into families, genera and species as well, assistance was fetched

19

from the reference keys and catalogues provided by Kaston (1978), Tikader and Malhotra

(1980), Tikader and Biswas (1981), Bringoli (1983), Tikader (1987), Barrion and

Litsinger (1995), Platnick (2004, 2009). After identification, all specimens were

deposited in the Museum, Department of Zoology & Fisheries, University of Agriculture,

Faisalabad. Most dominant species (adult specimens) were stored at -20 °C for the further

molecular studies.

Community ecology indices were applied by using the statistical software Ludwig

and Reynald (1988).

3.3 Utilization curve The relative partitioning of resources (prey groups) by a species is termed as

utilization curve (Ludwig and Reynald, 1988). Utilization curves were computed for each

of the nine synoptic species, based on observation data.

The No. of specimens constituted = % of total spiders (100 % = N = 488)

Utilization curves were used to estimate niche overlap and breadth in terms of

selective predation by spiders. Few species recorded in this study had less number of

observed cases (N

20

Where B is the Levins measure of niche breadth and Pi is the proportion of

individuals found using resource i. Often, these measures are standardized on a scale of 0

to 1 by using the formula:

BA = B-1/n-1

Where BA is the standardized niche breadth, and n is the total number of food

items for the species of interest.

An overlap value was computed for each of given species pairs. Values ranged

between “0” (no overlap) to “+1” (complete overlap) for each spider species. To quantify

the predation and habitat relationship value of niche breadth and overlap indices was

calculated from predation data of common species, foraging in different areas (part of the

plant) at different times.

3.6 Molecular studies The moleecular studies were performed in Molecular Biochemistry Lab.,

Department of Chemistry and Biochemistry, University of Agriculture Faisalabad. 3.6.1 DNA extraction

DNA extraction of the selected species was performed by three methods.

According to manual method (Cheung et al., 1993) in a 1.5 mL Eppendorf tube, spider

was lowered into liquid nitrogen for 8-10 s. It was homogenized with the help of a

sterilized plastic Eppendorf pestle, 500 µL chilled DNA buffer and 90 µL 5 % Sarcosyl

solution were added and then additional grinding was carried out to ensure complete

destruction of the tissues. Tubes were then incubated at 65 °C for one hour with

occasional mixing. Following incubation, the homogenized material was spun at 13,000

rpm for three minute to pellet the gross debris. The supernatant, containing the DNA, was

transferred to a fresh tube and precipitated in chilled isopropanol containing 90 µL of 10

M ammonium acetate. The pellet was washed in 70% ethanol, air dried for 30-40 min and

resuspended in 50 µL sterile water after heating at 60 °C for one hour. DNA was also

isolated by extraction kits from Fermentas and Promega following the manufacturer’s

protochols.

21

Solutions for the manual DNA extraction method were preapared as follows:

a. Lysis buffer reagents

200 mM Tris-HCL (pH 8.00) Tris base 12.11 g was mixed into a volume of deionized water, approximately 1/3 of the

desired volume of buffer, using a pH meter titrated with the solution of 1M hydrochloric

acid (HCl) until the correct pH is reached. Final volume was made up to 100 mL.

70 mM EDTA Na2EDTA (37.224 g) was dissolved in water. The pH was adjusted to 8.0 with 10

M NaOH the volume was made to 100 mL and autoclaved.

2 M NaCL 11.7 g of NaCL was dissolved in water and volume was made up to 100 mL by

adding distilled water.

20 mM Sodium metabisulphite 19.01 g of Sodium metabisulphite was dissolved in water, required volume was

made up to 100 mL by adding distil water.

b. 5 % Sarcosyl solution 5 g Sarcosyl was dissolved in water and made its volume up to 100 mL with the

addition of autoclaved distilled water.

c. 10 M Ammonium acetate 770.8 g Ammonium acetate was dissolved in 1 L distilled water to prepare the

required amount of the solution.

3.6.2 Confirmation of isolated DNA DNA isolation was confirmed by agarose gel electrophoresis. The samples were

run in 0.8 % agaros prepared in 1XTAE (Tris Acetate EDTA) electophoresis buffer. Fifty

microliters of ethidium bromide (0.05 g/100 mL) was added for staining. The DNA

samples of 5 µL containing 1 µL of 6X loading dye were loaded on the gel and

electrophorised at 80 V. The gels were documented on gel documentation system

(Syngene UK).

22

3.6.3 DNA quantification Absorbance of the samples was noted at 260 nm and 280 nm, on

spectrophotometer (GenQuant, Amersham Biosciences). DNA concentration was

determined by following formula:

Conc. of DNA (µg mL-1) = Dilution Factor × Abs. 260 × 50 (standard conc. of DNA)

As (standard conc. of DNA) Abs. 260 = 50 µg mL-1

3.6.4 Polymerase Chain Reaction (PCR) For polymerase chain reaction, optimized PCR cocktail concentrations were

utilized to amplify the sequence of cytochrome c oxidase 1 with different sets of primers.

The target region was amplified in 50 µL reaction mixture setup (Fermentas) as shown in

the Appendix 1.

Temperature cycling: Total 30 cycles were performed for each reaction. The PCR thermal regime was

set as follows:

Initial denaturation 94 °C for 5 min

Denaturation 94 °C for 1 min

Annealing 53 °C for 1 min

Extension 72 °C for 10 min

3.6.5 Primers designing To amplify the cytochrome oxidase 1 from above mentioned spiders, two standard

predesigned primer pairs were used in each PCR reaction (Folmer et al., 1994). Likewise,

to amplify the prey remnants within predators gut, seven different primer pairs were

selected.

Well-designed primer pairs of CO1 region for spider predators were used: Primer 1 (anti-sense)

5´-GTCAACAAATCATCATAAAGATATTGG

Primer 2 (sense)

5´-TACTCTACTAATCATAAAGACATTGG

23

Primer 1 (anti-sense)

5´-CCTCCTCCTGAAGGGTCAAAAAATGA

Primer 2 (sense)

5´-GGATGGCCAAAAAATCAAAATAAATG Functional primer pairs of CO1 region used for prey amplification were:


5´-AGTTTTAGCAGGAGCAATTACTAT

Primer 2 (sense)

5´-GCTAATCCAGTAAATAAAGG


5´-GAATAATTCCCATAAATAGATTTACA

Primer 4 (sense)

5´-TCAAGATAAAGGAGGATAAACAGTTC


5´-TATAGCATTCCCACGAATAAATAA

Primer 6 (sense)

5´-AATTTCGGTCAGTTAATAATATAG


5´-AGTTTTAGCAGGAGCAATTACTAT

Primer 8 (sense)

5´-TTAATWCCWGTWGGNACNGCAATRATTAT


5´-TACAGTTGGAATAGACGTTGATAC

Primer 10 (sense)

5´-AAAAATGTTGAGGGAAAATGTTA


5´-GTAAACCTAACATTTTTTCCTCAACA

Primer 12 (sense)

5´-TCCAATGCACTAATCTGCCATATTA

24


5´-TGATCAAATTTATAAT

Primer 14 (sense)

5´-GGTAAAATTAAAATATAAACTTC

3.6.6 DNA sequencing DNA from agarose gel was extracted by using DNA extraction kit from Qiagen.

Approximately 42 µL DNA was loaded on 1 % agarose gel until the loading dye traveled

two third volumes of buffer QG added to one volume of gel and incubated at 50 °C for 10

min with 2-3 min vortexing to dissolve the gel completely. One gel volume of

isopropanol was added to the sample, mixed and the sample was added to QIAquick

column. Centrifuged for one minute, flow-through was discarded and 1.5 mL of buffer

QG was added to QIAquick column and centrifuged for one minute. 0.75 mL of buffer

PE was added to QIAquick column for washing, incubated for five minutes and

centrifuged for one minute at 13,000 rpm. Flow through was discarded and centrifuged

the column for an additional one minute. QIAquick column was placed in a clean 1.5 mL

microcentrifuge tube and 50 µL of buffer EB was added to elute DNA and incubated for

5 min. After incubation, column was centrifuged for 1 min at 13,000 rpm. Purified DNA

was collected in a microcentrifuge tube and stored at -20 C. The purified fragments were

sequenced from Center of Excellence in Molecular Biology (CEMB), Lahore Pakistan.

3.6.7 Bioinformatic analysis All the anonymous sequences were subjected to the GenBank BLAST algorithm

for the sake of confirmation that all sequences were from Arthropoda. Sequences were

refined, FASTA formated and pairwise aligned by different online available software

pakages. Phylogenetic analysis was also constructed by Phylogeny Inference Package

(PHYLIP 3.67) (Felsenstein, 2005).

3.6.8 Deposits to GenBank® The entire purified new CO1 gene sequences were submitted to GenBank® to get

their accession numbers. NCBI (National Center for Biotechnology Information) helps to

keep the dataase as comprehensive, current and accurate as possible.

25

CHAPTER 4 RESULTS-Section I 4.1 Taxonomic and ecological studies 4.1.1 Predators recorded from December through April 2008

Predators along prey were captured during Dec. to Apr. from wheat fields of the

University of Agriculture, Faisalabad. The sampling was done for two years (2008,

2009). Predation data was obtained on daily basis and identified into their respective

families and genera. A total of 51 species, 20 genera and 7 families was recorded and

identified on seasonal basis. On monthly basis a total of 48, 117, 143, 153 and 116

spiders were captured with 577 predation evidences respectively. Maximum genera

belonged to family Araneidae Simon 1895 that comprised of Neoscona Simon 1864,

Argiope Audouin 1826, Cyrtophora Simon 1864, Leucauge White 1841; Araneus Clerck

1757, Cyclosa Menge 1866 and Gea C.L.Koch 1843 were recorded followed by family

Salticidae Blackwall 1841 with four genera Plexippus Koch 1849, Salticus Latreille

1804, Marpissa Koch 1846 and Phiddipus Koch 1846. On contrary to this the only

Tetragnatha Latreille 1804 belonged to family Tetragnathidae Menge 1866. Maximum

number of spiders related to family Lycosidae Sundevall 1833, genus Pardosa C.L.Koch

1848 (9) followed by Oxyopidae Thorell 1870 genus Oxyopes Latreille 1804 (7),

Araneidae, genus Neoscona (4) Tetragnathidae genus Tetragnatha (3), Salticidae genus

Plexippus Koch 1846 (2) were recorded (Appendix 2).

4.1.2 Predators recorded from December through April 2009 Overall spider predators comprised of 55 species, 20 genera, and 7 families

recoded on seasonal basis. Monthly 59, 158,168,169 and 112 were captured along their

prey respectively. Maximum number of genera belonged to family Araneidae while

genera of families Clubionidae and Tetragnathidae were found in least number. Overall

individuals were high in middle three months (Appendix 3).

The combined data of both years showed that Neoscona mukerji Tikader 1980

(78), Argiope pradhani Sinha 1951 (50), aemula Walckenaer 1841 (51), Oxyopes javanus

Thorell 1877 (58), Pardosa timida C.L.Koch 1848 (59), Hippasa olivacea Thorell 1887

(52), Tetraganatha javana Thorell 1890 (56) was found in very high abundance while N.

bengalensis Tikader & Bal 1981, G. subarmata Thorell 1890, C. venusae Barrion &

26

Litsinger 1995, O. tineatipes Simon 1864, O. hindustanicus Peters & Edmunds 1970, O.

campii Mushtaq & Qadar, 1999, P. myanmarensis Barrion & Litsinger 1995, P. mukundi

Tikader & Malhotra 1980, H. partita Cambridge 1876, L. poonaensis Tikader &

Malhotra 1980, L. tista Tikader 1970, T. nitens Audouin 1827 and T. okinawensis Strand

1907 were found ≤ 10 in captured specimens. Family Lycosidae was most populated

throughout the whole study period. It was 11.82% in February, 12.48 % in March and

10.58 % in April and with little bit difference in remaining months during 2008, which

makes 33.99 % of the total spiders. A steady increase in number were found during 2009

during January to March, which makes 12.90 %, 13.89 % and 13.97 % respectively

(Appendix 4).

4.1.3 Predator species dominance Information regarding the dominant species was documented, during five months

period, only species that were represented by four or more, later stage of development

was considered as dominant. Out of 1209 specimens, 488 constituted dominant synoptic

individuals. Table 4.1 provides the description of their abundance be as follows:

Neoscona mukerji That species was detailed during all months. It was evidenced by sudden

intensification and a gradual decline in integer of individuals throughout the entire study

period. Extreme value was documented in February 30 (38.4 %) and least number of

species was found in December 2 (2.5 %). As a whole it designed 78 (15.9 %) of the total

sample among dominant species.

Argiope aemula Same growing and falling trend was found in that species as it was observed

earlier. It constituted the maximum value 14 (27.4 %) in January and February as

compared to other months. Minimum number of specimens were recorded in April 7

(13.7 %) followed by December 3 (5.8 %). As a whole it formed 51 (10.4 %) of the total

sample among dominant species.

27

Leucauge decorata Maximum number of that species was found in middle two months 13 (28.8 %)

that was a stable increasing state among the other dominant species. An exact figure, as a

complete 45 (9.2 %) was obtained as compare to other species.

Plexippus paykulli Information regarding the dominance of P. paykulli was documented in an

accumulative mode throughout the study period. Whereas 4 (8.1 %) was the lowest value

recorded in December. As a whole it formed 49 (10.0 %) of total sample among

dominant species.

Cyclosa spirefera Information regarding the dominance of C. spirefera was described as an

increasing trend throughout the months with a sharp weakening in April 5 (12.5 %). It

comprised of about 40 (8.1 %) among completely dominant species.

Oxyopes javanus That species was reported throughout study period and its supremacy instituted a

gradual increase from first to last month. As a whole it formed 58 (11.8 %) among other

dominant species.

Hippasa olivacea Dominance of H. olivacea was remarkably plentiful 15 (28.8 %) in last month

April. The minimum figure was acquired 6 (11.5 %). It was also found throughout the

respective months. Its dominance as compare to other dominant species was documented

52 (10.6 %).

Pardosa timida That species was reported throughout complete study period and constituted the

greatest number of individuals in January 16 (27.1 %) with a stable state 11 (18.6 %) of

dominant species in middle months. As a whole it formed 59 (12.0 %) of the other

dominant reported species.

Tetragnatha javana The dominance of Tatragnatha javana increased in early months with a stable

state of individuals in the subsequent months. It was highest in January 15 (26.7 %) and

28

lowest in December 5 (8.9 %) than other recorded species. As a whole, it formed 56 (11.4

%) of the other dominant reported species.

The overall relative abundance of dominant predators was reported gradually in

elevation through initial months and then falls down (Table 4.1). Least seasonal based

abundance was documented in December 40 (8.10 %) followed by highest in February

125 (25.61 %) than other months. A comparative study showed February to be the most

favorable month to obtain a high extent of specimens.

Table 4.1 Relative abundance of dominant predators caught during 2008 and 2009

4.1.4 Insect prey recorded from December through April 2008

Prey obtained along predators was identified into orders, families and genera.

Overall, insect prey comprised of 30 genera, 23 families and 9 orders. A total number of

63, 72, 72, 71 and 63 insect specimens were captured from December, January, February,

March and April respectively. Prey related to genus Trialeurodes Cockerell 1902 (24)

followed by Aphis Linnaeus 1758 (22) documented a very high proportion throughout

study period. In addition, maximum number of families also belonged to order Diptera

(5) and maximum number of genera belonged to family Noctudiae Latreille 1809 (3),

Species Dec Jan Feb Mar Apr Tot

Tot/% Tot/% Tot/% Tot/% Tot/% Tot/% N.mukerji 2/2.5 16/20.5 30/38.4 20/25.6 10/12.8 78/15.9 A.aemula 3/5.8 14/27.4 14/27.4 13/25.4 7/13.7 51/10.4 L.decorata 5/11.1 8/17.7 13/28.8 13/28.8 6/13.3 45/9.2 P. paykulli 4/8.1 7/14.2 13/26.5 12/24.4 13/26.5 49/10.0 C.spirefera 3/7.5 9/22.5 10/25.0 13/32.5 5/12.5 40/8.1 O. javanus 3/5.1 9/15.5 13/22.4 15/25.8 18/31.0 58/11.8 H.olivacea 6/11.5 12/23.0 9/17.3 10/19.2 15/28.8 52/10.6 P.timida 9/15.2 16/27.1 11/18.6 11/18.6 12/20.3 59/12.0 T. javana 5/8.9 15/26.7 12/21.4 12/21.4 12/21.4 56/11.4 Total 40 106 125 119 98 488 % age 8.10 21.72 25.61 24.38 20.08 99.89

29

Muscidae Latreille 1802 (2), Curculionidae Latreille 1802 (2) Tenebrionidae Latreille

1802 (2), Thripidae Stevens 1829 (2) and Amphipterygidae Selys 1853 (2), (Appendix 5).

4.1.5 Insect prey recorded from December through April 2009 Overall, insect prey comprised of 22 genera, 19 families, and 8 orders. A total of

41, 43, 34, 33 and 17 specimens were captured from December to April respectively.

Maximum prey related to family Aleyrodidae Kirkaldy 1907 followed by Aphididae

Buckton 1879, Thripidae and Phoridae Malloch 1878 and order wise Homoptera (56)

was reported high in number as compare to Araneae (10), (Appendix 6).

The combined data of both years showed that out of 497 insect specimens Aphis,

Empoasca Walsh 1862, Trialeurodes, Aneurina Hebard 1935, Pheidole Westwood 1839,

Gryllotalpa Latreille 1802, Chrotogonus Serville 1838, Neoscona Simon 1864,

Caliothrips Daniel 1904 and Tanymecus Germar 1817 were very high in abundance

throughout years. Instead of that, March and April were reported with enriched insect

fauna during 2008 and 2009 (Appendix 7).

4.1.6 Prey taxa of nine synoptic spider species Information regarding to order wise prey families and genera used by nine

synoptic spider species along their utilization curve was noted as follow:

Prey utilization by Neoscona mukerji Order wise maximum number of prey utilization by N. mukerji was achieved from

Homoptera, Diptera and Orthoptera. This favorite food of spiders constituted over all the

1 % of total utilization curve. Most of the prey belonged to families Aphididae,

Aleyrodidae, Reduviidae Latreille 1807, Muscidae, Phoreidae, Acrididae MacLeay 1819,

Gryllotalpidae Saussure 1870, Noctuidae , Thripidae, Amphipterygidae and Araneidae

followed by genera Aphis (4), Gryllotalpa (4), Caliothrips (4) Trialeurodes, (3), Reduvis

(3), Aneurina (3), Chrotogonus (3), Agrotis (3), Coenagrion (3) and Neoscona (3)

respectively (Table 4.2).

30

Table 4.2 Order wise prey consumed by Neuscona mukerji and its utilization curve

Orders Families Genera Prey Utilization curve Homoptera Cicadellidae Empoasca 2

Aphididae Aphis 4 Aleyrodidae Trialeurodes 3 Reduviidae Reduvis 3 0.24

Diptera Muscidae Musca 3 Phoreidae Aneurina 3 Chloropidae Oscinella 2 0.16

Hymenoptera Cephidae Cephus 2 Formicidae Pheidole 1 Tenthredinidae Athalia 2 0.10

Orthoptera Acrididae Chrotogonus 3 Gryllotalpidae Gryllotalpa 4 0.14

Lepidoptera Noctuidae Agrotis 3 0.06 Coleoptera Curculionidae Alcidodes 2 0.04 Thysanoptera Thripidae Caliothrips 4

Anaphothrips 1 0.10 Odonata Amphipterygidae Coenagrion 3 0.06 Araneae Araneidae Neoscona 3 Oxyopidae Oxyopes 2 0.10 Total 9 18 19 50 1.00

31

Prey Utilization by Argiope aemula From the experimental findings, it was found that highly consumed prey by

Argiope aemula belonged to order Homoptera, Diptera and Araneae. This chosen food of

spiders established 0.94 % of total utilization curve. The most preferred prey fits to

family Aphididae, Aleyrodidae, Araneidae, Cicadellidae and Phoreidae monitored by

genera Aphis (5), Trialeurodes (5), Neoscona (4), Empoasca (3), and Aneurina (3)

respectively (Table 4.3).

Table 4.3 Order wise prey consumed by Argiope aemula and its utilization curve

Orders Families Genera Prey Utilization curve

Homoptera Cicadellidae Empoasca 3

Aphididae Aphis 5

Aleyrodidae Trialeurodes 5 0.31

Diptera Muscidae Musca 2

Phoreidae Aneurina 3

Chloropidae Oscinella 2 Dolichopodidae Dolichopus 2 0.21

Hymenoptera Cephidae Cephus 1

Formicidae Pheidole 2

Tenthredinidae Athalia 1 0.09

Orthoptera Acrididae Chrotogonus 2

Gryllotalpidae Gryllotalpa 1 0.07

Lepidoptera Noctuidae Agrotis 1

Sphingidae Acherontia 1 0.04

Coleoptera Curculionidae Alcidodes 1 0.02

Thysanoptera Thripidae Caliothrips 2 0.04

Odonata Amphipterygidae Coenagrion 1 0.02

Araneae Araneidae Neoscona 4

Oxyopidae Oxyopes 2 0.14

Total 9 19 19 41 0.94

32

Prey utilization by Leucauge decorata Maximum prey utilization rate of Leucauge decorata was documented with

Homoptera, Diptera, Orthoptera and Coleoptera that make a total 0.96 % utilization

curve. The highly consumed prey families reported were Aphididae Reduvidae,

Aleyrodidae, Gryllidae, Muscidae, Formicidae, Curculionidae and Thripidae followed by

genera Aphis (5), Reduvis (4), Trialeurodes (4), Acheta (4), Musca (3), Tanymecus (3)

Pheidole (3) and Caliothrips (3), respectively (Table 4.4).

Table 4.4 Order wise prey consumed by Leucauge decorata and its utilization curve

Orders Families Genera Prey Utilization Curve Homoptera Aphididae Aphis 5

Reduviidae Reduvis 4 Cicadellidae Empoasca 2 Aleyrodidae Trialeurodes 4 0.31

Diptera Syrphidae Sphaerophoria 2 Muscidae Musca 3 Phoreidae Aneurina 2 0.14

Hymenoptera Cephidae Cephus 1 Formicidae Pheidole 3 0.08

Orthoptera Gryllidae Acheta 4 Acrididae Schistocerca 2 Gryllotalpidae Gryllotalpa 1 0.14

Lepidoptera Noctuidae Earias 1 0.02 Coleoptera Curculionidae Tanymecus 3

Plutellidae Plutella 2 Tenebrionidae Mesomorphus 1 0.12

Thysanoptera Thripidae Caliothrips 3 Anaphothrips 1 0.08

Odonata Amphipterygidae Coenagrion 2 0.04 Araneae Araneidae Neoscona 2 0.04 Total 9 19 20 48 0.96

33

Prey utilization by Plexippus paykulli The total prey utilization curve of Plexippus paykulli was found to be 0.93 %.

Maximum feeding was relying upon the Homopterous, Dipterous, Hymenopterous and

Araneaous insects. The most favored prey families were Aphididae, Cicadellidae,

Aleyrodidae, Phoridae and Formicidae followed by genera Aphis (4), Empoasca (4),

Trialeurodes (3), Aneurina (3) and Pheidole (3), respectively (Table 4.5).

Table 4.5 Order wise prey consumed by Plexippus paykulli and its utilization curve


Homoptera Aphididae Aphis 4

Cicadellidae Empoasca 4

Aleyrodidae Trialeurodes 3 0.36

Diptera Phoridae Aneurina 3

Syrphidae Sphaerophoria 1 0.13


Formicidae Pheidole 3 0.13

Orthoptera Gryllotalpidae Gryllotalpa 1 0.03

Lepidoptera Sphingidae Acherontia 1 0.03

Coleoptera Curculionidae Tanymecus 1 0.03

Thysanoptera Thripidae Anaphothrips 2 0.06

Odonata Amphipterygidae Zygopteran 2 0.06

Araneae Oxyopidae Oxyopes 2

Araneidae Neoscona 2 0.13

Total 9 14 14 30 0.93

34

Prey Utilization by Cyclosa spirefera An intensive utilization of Chloropidae, Aphididae and Cephidae followed by

genera Oscinella (4), Aphis (3) and Cephus (3) was calculated that make of 0.96 %

utilization curve. While making a comparison of different orders, feeding by Cyclosa

spirefera was found maximum among the Homopterous, Dipterous and Hymenopterous

insects (Table 4.6).

Table 4.6 Order wise prey consumed by Cyclosa spirefera and its utilization curve


Homoptera Aphididae Aphis 3

Aleyrodidae Trialeurodes 2

Cicadellidae Empoasca 2

Reduviidae Reduvis 1 0.25

Diptera Chloropidae Oscinella 4

Phoridae Aneurina 1

Syrphidae Sphaerophoria 1 0.19


Formicidae Pheidole 2 0.16

Orthoptera Gryllotalpidae Gryllotalpa 2 0.06

Lepidoptera Noctuiodae Hypena 2 0.06

Coleoptera Curculionidae Tanymecus 1 0.03

Thysanoptera Thripidae Caliothrips 2

Anaphothrips 1 0.09

Odonata Amphipterygidae Coenagrion 2 0.06

Araneae Araneidae Neoscona 2 0.06

Total (9) 15 16 31 0.96

35

Prey Utilization by Oxyopes javanus The uppermost prey order exploitation was found in Homoptera, Araneae and

Diptera constructing utilization curve of 0.94 %. Maximum number of consumed prey

reported fit in families Aphididae, Aleyrodidae, Oxyopidae, Cicadellidae, Thripidae,

Araneidae and Phoridae followed by genera Aphis (6) Trialeurodes (6), Oxyopes (6),

Empoasca (4), Caliothrips (4), Neoscona (4) and Aneurina (3) respectively (Table 4.7).

Table 4.7 Order wise prey consumed by Oxyopes javanus and its utilization curve

Orders Families Genera Prey Utilization curve Homoptera Aphididae Aphis 6

Aleyrodidae Trialeurodes 6 Cicadellidae Empoasca 4 Reduviidae Reduvis 1 0.32

Diptera Chloropidae Oscinella 2 Phoridae Aneurina 3 Syrphidae Sphaerophoria 1 0.11

Hymenoptera Cephidae Cephus 2 Formicidae Pheidole 1 0.05

Orthoptera Gryllotalpidae Gryllotalpa 2 Acrididae Chrotogonus 2 0.07

Lepidoptera Noctuiodae Hypena 1 Agrotis 2 Earias 1 0.07

Coleoptera Curculionidae Tanymecus 1 Alcidodes 1 0.03

Thysanoptera Thripidae Caliothrips 4 0.07 Odonata Amphipterygidae Coenagrion 1

Zygopteran 1 0.03 Araneae Araneidae Neoscona 4

Oxyopidae Oxyopes 6 0.19 Total (9) 17 21 52 0.94

36

Prey Utilization by Hippasa olivacea A comparative study of order wise usage of prey by Hippasa olivacea belonged to

the Diptera, Hymenoptera, Coleoptera and Thysanoptera following a total utiliz

phylogenetic analysis and prey identification of spiders...

Documents