Diversity of aphid pests and assessment of molecular

The Journal of Zoology Studies

Vol. 3 No. 5 2016 Journalofzoology.com

Page 35

The Journal of Zoology Studies 2016; 3(5): 35-47

ISSN 2348-5914

JOZS 2016; 3(5): 35-47

JOZS © 2016

Received: 12-09-2016

Accepted: 29-09-2016

Faria Farhana Rain

Laboratory of Molecular

Entomology, Department of

Zoology, Jahangirnagar University,

Savar, Dhaka-1342, Bangladesh.

Abu Faiz Md. Aslam





Nayma Akter

Laboratory of Molecular Biology,

Department of Zoology,

Jahangirnagar University, Savar,

Dhaka-1342, Bangladesh.

Siddiqua Sultana Ringki




Savar, Dhaka- 1342, Bangladesh.

Abdul Jabber Howlader





Corresponding Author:

Abu Faiz Md. Aslam





Diversity of aphid pests and assessment of molecular

phylogenetic relationship based on mitochondrial gene sequence

Authors: Faria Farhana Rain, Abu Faiz Md. Aslam, Nayma Akter, Siddiqua Sultana Ringki,

Abdul Jabber Howlader

Abstract

An experiment was conducted to determine the aphid pests’ diversity in Jahangirnagar

University Campus from January to December, 2015. Total ten aphid pests were identified viz,

Acyrthosiphon pisum, Aphis craccivora, Aphis fabae, Aphis gossypii, Aphis nerii, Brevicoryne

brassicae, Hyadaphis coriandri, Lipaphis erysimi, Macrosiphum euphorbiae, Myzus persicae..

Exploration of aphid pests showed that they caused serious damage to agricultural crops by

attacking different part of plants. Seasonal abundance of aphid pests showed that they had close

relation with meteorological factor. From the last of September, aphid populations were found

to decrease gradually mainly due to rain and appearance of many natural enemies in the crop.

Along with diversity study, phylogenetic relationship of aphid pests was constructed based on

mitochondrial COI gene. In sequence alignment, out of ten aphids Acyrthosiphon pisum showed

non conserve region than rest. Analysis of interspecific genetic divergence exposed the genetic

range of aphid pests was 0.05-0.11. For confirmation of origin and evolution of aphid pests,

phylogenetic tree and haplotype analysis was performed. Both Neighbor-Joining and Maximum

likelihood methods for tree construction and haplotype analysis showed My. persicae was

genetically most diverge among all other studied aphids. Perhaps the control program for one

aphid species will be applicable for other aphid pests because they originated from common

ancestor and they were closely related to each other. This information would be helpful in

Integrated Pest Management program for successful control of aphid pests in Bangladesh.

Keywords: Aphid; diversity; Seasonal abundance; COI gene; Phylogenetic analysis; Haplotype

1. Introduction

Aphids (Hemiptera: Aphididae) are the most destructive and widely distributed herbivorous

insects accounting more than 4,300 described species [3, 4, 15]

. They attack the crops and cause

harm by imbibing the sap from different parts of the plant and lessen the market price [39]

.

Aphids are the most common vector of plant viruses [1]

. This pest account for yield losses

ranging from 37 to 90% [28]

. Most plants have one or more species of aphid that have been

known to feed on it. Through their huge reproductive ability, aphids can cause severe damage to

crops. At present, the main aspect of the aphid control program is based on the use of chemical

insecticides. Continuous usage of chemicals has some disadvantages like pest resistance,

environmental contaminations and sometimes outbreak of secondary pests [27, 29, 44]

. Some aphids

have developed resistance to at least seventy different synthetic compounds [11, 37]

. These

entomological backlashes have bound the scientists to be concerned with environment friendly

pest management programs [19]

.



Page 36

IPM requires an understanding of the ecology of the

cropping system, including that of the pests, their

natural enemies, and the nearby environment. Timely

monitoring of pest populations gives information

needed to decide if and when to apply a specific

control measure for optimal effectiveness. There are

scarcities of research on aphid management in

environmentally safe manner, more specifically using

seasonal information and natural enemies in

Bangladesh. In Bangladesh, aphid fauna are mostly

unrecorded [21]

. This situation led to design a research

to know some valuable information about aphid

diversity, its nature of damage, seasonal abundance and

relation of climate with the pest incidence.

Understanding of aphid phylogeny will eventually help

to make the proper pest control strategy. Attempt to

reconstruction of aphid Phylogeny has been

accomplished around 20 years ago but ended with

some conflict [18, 45]

. The reason of conflict was

insufficient phylogenetic data. Recently some efforts

have been taken to understand the aphid phylogenetic

relation based on Mitochondrial COI gene. COI gene

have only maternal transmission and they emerge more

quickly than nuclear genes and comprise much higher

potential coding region than nuclear genomes [7, 36]

.

COI gene has been widely used because it is the

standard tool of identifying the organisms and also

helps in understanding phylogenetic analysis [8]

. Aphid

classification at various levels has been attempted

using morphological characters, host-plants

associations and life cycles [3, 18]

. However,

morphological studies very often tend to be uncertain.

The phylogenetic analysis was also performed to know

the relationship among aphid. This finding will help in

pest control program in control measure of aphid. Thus

our farmer will able to produce quality agricultural

products in increased number in an environmental

safety manner and thus could reach the millennium

development goals.

2. Materials and Methods

2.1 Study area of aphid faunal survey and

preparation of experimental plots

Aphids were collected from different vegetables,

ornamental trees, shrubs, flowers, and other crops that

are economically valuable from different parts of

Dhaka city, capital of Bangladesh (23° 42' N, 90° 24'

E)from January – December, 2015. Home garden,

commercial nurseries, different crop fields, botanical

garden and Insect Rearing and Experimental Station

(IRES) was main focusing area to collect aphid.

For conducting this research few small plots of

different crops (bean, eggplant, cabbage, and mustard)

were prepared in the Insect Rearing and Experimental

Station (IRES) of Jahangirnagar University in addition

to observation in other parts of the campus and its

surroundings. In the small plots any chemical fertilizer

or insecticide were not used to keep the insects

undisturbed. Data obtained from IRES were used to

determine seasonal abundance of the pest. The

ecological condition supports the prey predator

relationships because this area is free of chemical

usage.

2.2 Collection and identification of aphid

Aphids were collected from the host plant using soft

camel hair brush and preserved into a small vial for

further studies in the laboratory of Molecular

Entomology, Wazed Miah Science Research Center,

Jahangirnagar University, Savar, Dhaka. Aphids were

identified using proper keys [5, 13, 14, 26, 33, 43]

.

2.3 Survey on the host of aphid and observe nature

of damage

Different types of plants were checked to find aphid

pest. Different aphids attack different plant in different

time of year. This is why all economic important plants

were checked throughout the year. Plants, where

aphids were found to attack as pest were considered as

host plant.

2.4 Monitor seasonal abundance of the pest

Aphids were checked throughout the cropping season

to check its seasonal abundance for a particular crop.

Aphids were count every week. At least 5 plants were

taken randomly. Then aphids of 10 randomly taken

leaves of a plant were counted using a magnifying

glass. Total number of aphids was then averaged to

count its abundance. Both wings and wingless were

taken into consideration while count. In the present

experiment bean aphid (Aphis fabae) of bean plant

(Lablab perpureus) was target pest.

2.5 Meteorological data

Records for mean relative humidity, mean air

temperature (°c), and total rainfall (mm) during the

sampling period in JU campus were collected from the

Department of Geography, Jahangirnagar University.

2.6 Collection of data for phylogenetic analysis

COI gene sequence of ten (10) aphid pests was

collected from National Center for Biotechnology

Information (NCBI) for their molecular and

phylogenetic analysis [22]

(Table 1).



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Table 1: Information of Aphid pests COI gene seuences

Name of Species GenBank Accessions (COI)

Acyrthosiphonpisum KR579669.1

Aphis craccivora KR084999.1

Aphis fabae GU324636.1

Aphis gossypii KR085028.1

Aphis nerii KR044621.1

Brevicorynebrassicae KJ814964.1

Hyadaphiscoriandri DQ499054.1

Lipaphiserysimi KJ814967.1

Macrosiphumeuphorbiae KR045247.1

Myzuspersicae KJ814969.1

2.7 Sequence alignment, Genetic distances and

Phylogenetic analysis

COI gene sequences were aligned using ClustalW

algorithm with the help of MEGA tools (version 6)

with gap opening penalty 15, gap extensions penalty

6.66, transition weight 0.5 and delay divergent cutoff

30% [24, 38]

. BioEdit Sequence Alignment Editor

Version 7.1.3help to produce multiple sequence

alignment image [16]

.

Kimura’s two parameter (K2P) genetic distances was

carried out using MEGA 6.The evolutionary history

was inferred using both the Neighbor-Joining (NJ) and

maximum likelihood (ML) tree method based on

MEGA 6 [35, 41]

. The percentage of replicate trees in

which the associated taxa clustered together in the

bootstrap test (1000 replicates) was shown next to the

branches [12]

.The tree was drawn to scale, with branch

lengths in the same units as those of the evolutionary

distances used to infer the phylogenetic tree.

Haplotypes were constructed using the program Pop art

1.7 based on TCS network [10]

. Haplotype identifies the

mutation rate among the aphid species.

3. Result

Different species of aphids that were collected and

identified following proper keys have been mentioned

in table 2. Different agriculturally important hosts of

the aphids were also recorded, identified and

mentioned in the same table. A total of ten agricultural

important aphids were identified up to species level

from the study area. Their short identification

procedure has been described in the table 2. All

identified species were pest of different agricultural

important crops. Most of the aphids were found as pest

of more than one plant due to their polyphagous habit.

Table 2: Different aphids and their agricultural host plants found in the JU campus

Common

name Scientific name Short taxonomic description of the pest Host plant

Pea aphid Acyrthosiphon pisum

Pale green with black legs; 4-5mm long, green, with a

shiny appearance; antennae are pale green or brown,

but always have a dark band at the joint of segments

III and IV.

Pea

Cowpea

aphid Aphis craccivora

Velvety black, with a distinct wax cover; Abdomen:

aptera - dorsum with black patch which is variable in

size; Alata - dorsum with black bands on all segments.

Pea, bean

Bean aphid Aphis fabae

Black or very dark brown species with a variable

abdominal sclerotic pattern - confined to abdominal

tergites 6-8 in smaller apterae but broken bands

present in larger ones; siphunculi and caudal are dark.

Bean, carrot,

lettuce, spinach

Cotton aphid Aphis gossypii

Wingless are generally dark green or blue-green with

dusky siphunculi; Abdomen: aptera - no dorsal

markings, reticulated pattern on dorsum; alata - no

black, dorsal bands on segments I to III and usually

none on IV and V.

Cucumber,

pumpkin,

eggplant, papaya



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Milkweed

aphid Aphis nerii

Wingless forms are yellow with black cornicles,

antennae, legs, and cauda.

Oleander,

milkweed

Cabbage

aphid Brevicoryne brassicae

Gray green with a powdery, waxy covering;Abdomen:

aptera - broken, dark, dorsal bands on all segments;

alata - broken, dark, dorsal bands on all segments.

Radish, cabbage,

cauliflower,

broccoli

Coriander

Aphid Hyadaphis coriandri

Yellow-green, dusted with greyish wax; short, dusky,

slightly swollen, cornicles that are about twice as long

as wide.

Coriander

Mustard

aphid Lipaphis erysimi

Yellowish green, gray green or olive green with a

white waxy bloom covering the body.

Mustard, broccoli,

cabbage, radish,

tomato

Potato aphid Macrosiphum euphorbiae

Pink or green; pear-shaped; long, dark

antennae;abdomen: no dorsal markings

Sweet potato,

cabbage, bean

tomato, eggplant

Green peach

aphid Myzus persicae

Wingless adult aphids vary from green to pale yellow;

winged adults are green with black or dark brown

markings on their abdomens.

Brinjal, potato,

cabbage, carrot,

tomato, papaya

3.1 Intensity of infestation in relation to time

Large aphid populations were found to cause curling,

yellowing, and distortion of leaves and stunting of

shoots. They were also found to produce large

quantities of a sticky substance known as honeydew,

which often turns black with the growth of a sooty

mold fungus.

Aphids were found to attack almost all parts of plant

viz., leaf, twig, flower and fruit (Fig 1). Direct damage

caused by aphids feeding caused yield losses of more

than 50% in some susceptible bean or mustard varieties

(data not presented in this paper). Observation revealed

that direct feeding damage occurs when colonies of 30

or more aphids were developed on individual growing

tips. The degree of damage depends on the varietal

susceptibility, the growth stage of the crop, the

percentage of plants infested, the number of aphids per

growing tip, and the duration of the infestation.

Different time of the year was not same for aphid

infestation. Aphid infestation was observed heavily in

the months January, February, and December to most

crops. But some early susceptible crop variety was

found to attack in the mid time of year.

Fig 1: Damage caused by aphids: a) Aphids sucking sap of bean twig b) stunted growth of bean c) stunted growth of

broccoli d) Leaf curling of egg-plant e) Aphid attack f) Egg-plant flower

3.2 Seasonal abundance of bean aphids

Seasonal abundance of bean aphid into different parts

of plants, viz., leaf, flower and bean were observed in

some experimental plots of IRES. Aphids were found

to attack vegetative part of bean (Aphis fabae) on mid-

August. Mostly newly emerged leaf and twig are their

choice place to infest. They were not found in mature

leaves. When the flower emerged or fruit comes out,

pests were found to attack those parts also in addition

to leaf during September to October, 2015.Fig. 2 shows



Page 39

the seasonal abundance of this pest in bean over the

cropping season. From the last of September

population were found to decrease gradually mainly

due to rain and appearance of many natural enemies in

the crop. In month of September, aphid infestation was

most and in the month of October aphid population

decreased slowly.

Fig 2: Seasonal abundance of bean aphids at IRES in different parts of the plant (values indicate mean±sem

(nymphs + adults) per 10 leaves.

3.3 Relation of Aphid with Environmental factors Meteorological data were recorded with a view to

check its relationship with the aphid pests of JU

campus. Mean relative humidity (%), mean air

temperature (°c), and total rainfall (mm) of the study

area throughout the year has been mentioned in the Fig.

3. It was found from the figure 1 and 2 that aphid has a

relationship with weather parameters. Aphids were

abundant in the month of January, February and

December when temperature was low (18.92 ºC, 21.54

ºC, 20.15 ºC respectively), coupled with light rain.

Aphids were least in May to August when temperature

and rain was maximum. Heavy rain was found to wash

away these pests.

Fig 3: Mean monthly temperature, humidity and rainfall recorded from Jahangirnagar University campus during

January-December, 2015.



Page 40

3.4 Multiple sequence alignment analysis

Multiple sequence alignment was presented by

showing the non-conserve region in letter and 70%

identical or conserved region by dots (Fig 4).

Acyrthosiphon pisum showed large amount of non-

conserved region than other aphid species. Other

species showed high conserved region among them.



Page 41



Page 42

Fig 4: Multiple sequence alignment of aphid pestsbased on COI gene sequences performed by Bioedit. Dot denotes

the conserved region.



Page 43

3.5 Genetic distance

Interspecific genetic divergence range of aphid

pestswas 0.05-0.11.My.persicaeshowed highest (0.11)

pairwise distance than rest. L.erysimi and

M.euphorbiae both showed lowest (0.05) pairwise

distance among studied aphid species.

Table 3: Interspecific K2P sequence divergence at the COI barcode region among the aphid pests

1 2 3 4 5 6 7 8 9 10

Acyrthosiphonpisum -

Aphis craccivora 0.08 -

Aphis fabae 0.09 0.06 -

Aphis gossypii 0.10 0.08 0.07 -

Aphis nerii 0.10 0.08 0.08 0.08 -

Brevicorynebrassicae 0.06 0.09 0.08 0.08 0.08 -

Hyadaphiscoriandri 0.07 0.09 0.09 0.10 0.09 0.06 -

Lipaphiserysimi 0.08 0.09 0.09 0.08 0.08 0.05 0.08 -

Macrosiphumeuphorbiae 0.05 0.09 0.10 0.10 0.09 0.06 0.07 0.08 -

Myzuspersicae 0.10 0.11 0.10 0.10 0.10 0.09 0.08 0.09 0.09 -

3.6 Phylogenetic analysis In both phylogenetic trees (Maximum likelihood and

Neighbour-Joining tree) grouped the ten aphid species

into three clusters. In neighbor joining tree,

A.craccivora, A.fabae, A.gossypii and A.nerii were

clustered in one group. B.brassicae and L.erysimiwere

grouped within same cluster and showed close

relationship. Ac.pisum, M.euphorbiae, H.coriandri and

My.persicaeclusterd into another group and showed

close relationship. Same results were also shown in

maximum likelihood tree. According to branch length,

My.persicaewas genetically most distant species from

the common ancestor observed in both tress (Fig 5).

Fig. 5: Evolutionary relationship among ten aphid species was constructed using MEGA6 software. In (a): Neighbor

joining tree, the optimal tree with the sum of branch length = 0.36257569 is shown. The percentage of replicate trees

in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer

the phylogenetic tree. In (b): Maximum likelihood tree, the evolutionary history was inferred by based on the

Kimura 2-parameter model. The tree with the highest log likelihood (-2213.9018) is shown. The percentage of trees

in which the associated taxa clustered together is shown next to the branches. The bar at the bottom 0.01 is a scale

for the genetic change.



Page 44

3.7 Haplotype

Haplotype analysis of mitochondrial COI gene of aphid

showed high genetic diversity among them. A.

craccivora and A. fabae separated from its immediate

common ancestors by 23 and 14 mutational steps.

Again these two species were separated from A.

gossypii by 9 mutational steps. L.erysimi, B.brassicae,

A. nerii, Ac. pisum, M. euphorbiae, H. coriandri and

My. persicae were diverged from its immediate

common ancestors by 16, 11, 32, 17, 15, 15 and 30

mutation number respectively. My.persicae showed

highest amount of mutated sites among other aphid

species and genetically distant from its common

ancestors by 30 mutational numbers (Fig 6).

Fig 6: Mitochondrial COI gene haplotype analysis of aphid species constructed by Popart 1.7 based on TCS

network. Red circles represent the haplotype and small yellow circles represent the immediate common ancestors.

Mutational steps are presented by hatch marks and numbers.

4. Discussion

A total of 10 agricultural important aphid pests have

been recorded from Jahangirnagar University and its

surroundings during this study period (Table 2). In

Bangladesh, only about 30 aphid species under 20

genera infesting 58 host plants have been listed in

national encyclopedia of Bangladesh, Banglapedia.

This represents less than 0.75% of the world and

2.94% of the Oriental and 4.59% of Indian aphid fauna [21]

. Bean aphid and Mustard aphid was previously

described for their different management aspects

elsewhere in Bangladesh [9, 20, 40]

. Bhagat described 31

aphid species, distributed over 20 genera, damaging 31

species of agricultural crops of 12 different plant



Page 45

families in India [2]

. Rafi et al. (2010) found fourteen

different winter vegetables and 13 ornamental plants

attacking aphid in Aligarh region, Uttar Pradesh of

India [33]

. Globally, more than 4,000 species of aphids

of 10 families have been described, some 250 of which

are pests of crops and ornamental plants [13, 43]

.

Present study reported abundant aphids in the month of

January, February and December when temperature

was relatively low coupled with foggy weather. Aphids

were least in May to August when temperature and rain

fall was maximum (Fig 2, 3). When we consider Bean

aphid (Aphis fabae), the infestation was found mostly

high in the month of September. Life of cycle Lablab

perpureus needs three month (August, September,

October) for its vegetation, flowering and fruiting.

Present findings were somewhat related to similar

research of other researchers. Hasan et al. (2009)

observed that aphid populations were correlated with

the variation of weather parameters in mustard

ecosystem. Aphid infestation increased because of

trace amount of rainfall, dew point coupled with very

low sunshine hours. Mustard aphid population declined

after end of January [17]

. Kumar et al., (1997) reported

that average temperature of 18.06 0C (Maximum

22.810

and Minimum 13.31 0C) under the influence of

high relative humidity with the range from 80.71% to

86.5% provided conductive conditions for aphid

incidence. Among the environmental factors, the

maximum temperature and dew point positively

correlated with aphid population [25]

. Aphids reproduce

quickly when conditions are right. During the spring

and summer females give birth to live offspring (not

eggs) which are all female. At the end of the summer

males are produced, and mated females lay eggs that

hatch the next spring [23]

(Davidson and Antonelli,

2003).

To know the aphid phylogeny relationship, we

obtained the COI gene sequences of ten aphid species

from NCBI(www.ncbi.nlm.nih.gov). Except

Acyrthosiphon pisum, all species of aphid showed high

conserve region (Fig 4). Interspecific K2P genetic

divergence range of aphid was 0.05-0.11. Among

studied all aphid species, My. persicaeshowed highest

(0.11) pairwise genetic distance than rest (Table 3).

Some phylogenetic experiments have been performed

to solve the mysterious problem of cryptic species of

aphid [8, 34]

. Previous works on aphid describing three

distinct groups with either viviparous, oviparous and

parthenogenetic groups [45]

. More recent efforts have

applied DNA sequence data to the reconstruction of

Aphididae phylogeny [30]

. In most cases, molecular

tools have proved important for species identification

and delimitation in aphids [6, 14, 31, 32]

. To avoid the

problems associated with the presence of too many

mutations or insertion–deletion events, scientists now

chose to work on a well-conserved coding region of

mitochondrial DNA. Thao et al., 2004 experimented

upon aphid COI gene to understand the phylogenetic

relationship [42]

. Rebijith et al., 2013 experimented on

aphid pest and Brevicoryne brassicae, Hyperomyzus

carduellinus and Brachycaudus helichrysi revealed that

the intraspecific and interspecific distances ranged

from zero to 3.8% and 2.31 to 18.9%, respectively [34]

.

In our present study, A. crassivora, A. fabae, A.gossypii

and A. nerii showed monophyletic clade and closely

related to each other in neighbor joining tree. B.

brassicae and L. erysimi belonged to the same cluster.

The highest bootstrap value (88%) was observed in

cluster of Ac. pisum and M. euphorbiae(Fig 5. a). In

maximum likelihood tree, highest bootstrap value

(86%) was observed in the cluster of A. nerii, A.

gossypii, A. craccivora and A. fabae (Fig 5.b). Ac.

pisum and M. euphorbiae were closely related to each

other. B. brassicae and L. erysimi were in

monophyletic clade showing genetic close relationship.

The haplotype analysis was done by TSC network and

the results revealed that My. persicae was genetically

most distant from its common ancestors by highest (30)

mutation number (5) ( Fig 6).From all phylogenetic

analysis, it can be conclude that My. persicae was

genetically most diverge among all other studied

aphids.

5. Conclusions

Aphids are considered one of the most injurious pests

of crops and economically important plants of

agriculture and horticulture. Proper pest control

program needs the biology, ecology and phylogenetic

data to pests. In this study, seasonal abundance of bean

aphid pest over the cropping season exposed that

population decreased gradually from the last of

September. Aphid attacked different parts of the plants

specially fruiting part. Phylogenetic data revealed that

all aphid pests are closely related and arose from a

common ancestor. Thus making control program of

one aphid pest will be applicable for other aphid pests

because they share close phylogenetic relation. Further

detailed studies on different aspects of aphid IPM will

help the farmers to get rid of this pest in an

environmental safety manner.

6. Acknowledgement

This work was partially supported by grants from

Higher Education Quality Enhancement Project (CP-

3424), a project of University Grants Commission of

Bangladesh and Ministry of Education, Bangladesh.



Page 46

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Diversity of aphid pests and assessment of molecular