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4 Role of Taxonomy in Management of Mealybugs

4 Role of Taxonomy in Management of Mealybugs

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4 Taxonomy

there have been more than five major outbreaks

of mealybugs causing alarming damage to crops,

as a result of accidental introduction.

The pink cassava mealybug, Phenacoccus

manihoti Matile-Ferrero, appeared on cassava in

Africa in 1973 and soon spread throughout the

whole cassava belt. To locate the source of this

mealybug and to compare it with a morphologically similar species, which causes almost identical damage to cassava in northern Brazil and

Guyana, required a considerable amount of time.

Specimens from Africa and northern South

America on the microscope slides showed wide

variation in characters, and the method adopted

to ascertain any limits to this variation required

rearing cultures in the laboratory at different temperatures. This method normally induces wide

morphological variation in mealybugs, helping to

determine the limits of environmentally induced

variation (Cox 1982; Cox and Williams 1981).

The knowledge that the species in Africa was

pink-bodied and uniparental and the species from

northern Brazil and Guyana was yellow-bodied

and biparental (later described as Phenacoccus

herreni Cox and Williams) was obscured initially

because the specimens used for this study were

dead and had been preserved in spirit, obscuring

the body color. When the two species could be

identified satisfactorily on the microscope slides,

it became apparent that the pink cassava mealybug, P. manihoti, was present in Paraguay and

Bolivia (Williams et al. 1981); hence, a search for

natural enemies could be implemented there. The

introduction of the parasitoid Apoanagyrus lopezi

(De Santis) from South America to Africa and the

success of the biological control program against

P. manihoti were well documented by

Neuenschwander and Herren (1988) and Herren

and Neuenschwander (1991). Thus, the taxonomic information can be retrieved in case the

mealybug introductions originate from this area.

Phenacoccus manihoti remains a threat to the

cassava areas of southern Asia, as does the yellow

cassava mealybug, P. herreni, which still causes

problems in South America. Reduction of P. herreni populations is now under way, mainly

through the introduction of the parasitoids

Apoanagyrus diversicomis (Howard) and


Acerophagus coccois Smith (Bento et al. 1999).

The most trenchant point concerning the parthenogenetic species P. manihoti is that an outbreak

could occur in southern Asia with the accidental

introduction of just a single immature specimen.

Following the introduction of the cassava mealybug into Africa, another introduced mealybug

appeared in West Africa in 1981–1982, causing

extensive damage to fruit trees including mango.

This mealybug was initially identified as an

undescribed species already known from India

and Pakistan and was later described as

Rastrococcus invadens Williams (Williams 1986).

This species is usually scarce in some parts of

India because it is controlled by the natural enemies (Narasimham and Chako 1988); the introduction of the encyrtid Gyranusoidea tebyi Noyes

from India to West Africa and its swift control of

the mealybug are hailed as another biological

control success (Neuenschwander et al. 1994).

Another mealybug species was introduced

accidentally to the Caribbean area in 1993–94

and has since then spread beyond, eventually

reaching USA. This damaging species was rapidly identified by taxonomists as Maconellicoccus

hirsutus (Green); its biological control was

described in detail by Kairo et al. (2000), with

discussion of the costs and benefits. M. hirsutus

is widely distributed throughout the southern

Asia, Africa, and other parts of the Old World

including Australia, and is still causing damage

in some parts of India. The introduced natural

enemies, mainly the parasitoids Anagyrus kamali

Moursi (already known in the Old World) and

Gyranusoidea indica Shafee, Alam and Agarwal

(collected in Egypt), and the predator

Cryptolaemus montrouzieri Mulsant, have

brought the mealybug under control. In response

to this outbreak, an identification manual for the

area (Watson & Chandler 1999) and a taxonomic

study of all the instars of M. hirsutus (Miller

2002) were produced.

Yet another mealybug is causing concern in

the Caribbean area Paracoccus marginatus

Williams and Granara de Willink, described from

Mexico and parts of Central America as recently

as 1992, has become a serious pest in the

Caribbean islands, where it attacks numerous

M. Mani


Table 4.2 List of mealybug species, correct identity of which led to a successful biological control

Mealybug species

Phenacoccus manihoti Matile-Ferrero

Phenacoccus herreni Cox and


Rastrococcus invadens Williams

Maconellicoccus hirsutus Green

Paracoccus marginatus Williams and

Granara de Willink

Paracoccus marginatus Williams and

Granara de Willink

Country of accidental



South America

Introduced parasitoid for classical biological


Apoanagyrus lopezi (De Santis)

Apoanagyrus diversicornis (Howard)

West Africa


Caribbean islands

Gyranusoidea tebyi Noyes

Anagyrus kamali Moursi

Acerophagus papayae Noyes and Schauff

Pseudleptomastix mexicana Noyes and Schauff

Anagyrus loecki Noyes and Menezes

Acerophagus papayae Noyes and Schauff

Pseudleptomastix mexicana Noyes and Schauff

Anagyrus loecki Noyes and Menezes


plant species, especially papaya (Carica papaya).

The mealybug has now reached the southern

USA. A search for natural enemies in Mexico

(Becker 2000) located three parasitoids that are

now in use in controlling the mealybug. An offshoot of the biological control program has been

a detailed study of all the instars of P. marginatus

by Miller et al. (2005). The mealybug had

affected Carica papaya and several other plants

in Guam, Sri Lanka, Palau, India; in all countries,

the species was rapidly identified as P. marginatus by taxonomists facilitating quick introduction of the parasitoids.

Table 4.2 shows a list of mealybug species,

which were introduced in some countries, and the

parasitoid, whose correct identity led to a successful classical biological control.

The examples mentioned above show that the

new pest species that may have escaped detection

at quarantine inspection of imported plant

material can be quickly recognized by the taxonomists and accurately identified. The taxonomists

can also suggest the correct area of the origin of

the pest and report whether any existing specimens in slide collections were parasitized, so that

the precise collection localities can be searched

for natural enemies for use in classical biological

control. This information requires access to

important reference collections of insects and to

the relevant taxonomic literature. The above field

guides and taxonomic information are to be

referred for the quick tentative identification up

to the species level. If the specimen does not

come under the existing keys, it may be named as

a new species.

Acknowledgement Dr. Sunil Joshi, Principal Scientist,

NBAIR, Bangalore is acknowledged for providing



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Molecular Identification

of Mealybugs

K.B. Rebijith, R. Asokan, and N.K. Krishna Kumar

Insects are the numerous life forms that have captured the attention of human beings since ancient

times. In the same context, proper classification

and identification of life forms has been a challenge, and a plausible method of classification

was established by Carlous Linnaeus, a Swedish

botanist who published Systema Naturae in 1758.

However, the Linnaeus system of classification

was not based on evolutionary relationships

among the target groups. Later, Darwin’s “The

Origin of Species” in 1859 changed the way life

forms were classified, where the identification,

description and explanation of the diversity of

the organisms had come to be known as systematics. According to Mayr and Ashlock (1991),

systematics is the scientific study of the kinds and

diversity of organisms, and any and all relationships among them; taxonomy, on the other hand,

was the theory and practice of the classification

of organisms. It took 200 years for taxonomists

to describe the 1.7 million species on the earth,

which is only 10 % of the total number of species

estimated. In this context, identification of insects

K.B. Rebijith • R. Asokan (*)

Division of Biotechnology, Indian Institute of

Horticultural Research, Bangalore 560089, India

e-mail: rebijith@gmail.com; asokaniihr@gmail.com

N.K.K. Kumar

Indian Council for Agricultural Research,

New Delhi, India

e-mail: ddghort@gmail.com

has been a monumental task which calls for the

availability of more specialists and funding. But

with the dwindling interest in taxonomy and fund

availability, the classification and identification

of various life forms, particularly insects, has

been a major challenge to the scientific community. With the advent of molecular biology and

molecular tools, the identification of life forms,

including insects, has become quick, precise and

easy. The development of species-specific markers enables even a non-specialist to identify

insects to the species level.


Methods of Classification

and Identification


Linnaean System

Taxonomists assess the physical characteristics

that a set of species shares and selects the most

representative species to be the ‘type’ for each

genus, and the most representative genus to be

the type of the family and so on. Individual specimens are deposited in museums to serve as a reference for that species and genus. When new

species are found with similar traits, they are categorized as part of a known species as a new species, or as a new genus, depending on how closely

the new specimens resemble the type. The reliance of types results in dramatic changes if a

taxonomist re-evaluates a group and decides that

© Springer India 2016

M. Mani, C. Shivaraju (eds.), Mealybugs and their Management in Agricultural

and Horticultural crops, DOI 10.1007/978-81-322-2677-2_5


K.B. Rebijith et al.


some members do not belong and suggest that the

group name must be changed.



During the 1980s, another classification method

called cladistics, which is based on the evolutionary histories of organisms, was proposed. This

method is based on phylogeny, whereas the

Linnaean system is not.



In this system, the genus name is removed, and

species name is shortened and hyphenated with

their former genus name or given numeric



Shortfalls in Morphological


Mealybugs (Hemiptera: Pseudococcidae) are the

major pests in a wide range of agricultural crops

as well as ornamental plants worldwide (Millar

2002; Miller et al. 2002, 2005). These sapsucking insects have been studied intensively for

decades because of the economic losses they

cause to agriculture through direct physical damage to crop plants, as well as by vectoring many

plant pathogenic viruses, which in turn decreases

yield quality (Meyer et al. 2008 and Nakaune

et al. 2008). The family Pseudococcidae consists

of more than 2000 described species in 270 genera (Downie and Gullan 2004). Current estimates

suggest that the earth may have anywhere from

10 to more than 40 million species of organisms,

but only about 1.7 million of them have actually

been described. It includes over 7,50,000 insects,

and it took 250 years for taxonomists to categorize all 1.7 million species, which comprise only

10 % of the total species on earth (Hebert and

Gregory 2005). Classifying the remaining 90 %

of the unidentified organisms will require more

time and expertise of taxonomists to complete

this monumental task. Economic development

and increased international commerce are lead-

ing to higher extinction rates and the introduction

of invasive species of pests. Therefore, there is a

need for faster species identification and information about their biodiversity for conserving

them before they vanish from the face of the

earth. Undoubtedly, the contribution of morphological taxonomy is enormous, but it also has

some drawbacks, such as the following:

• Incorrect identification due to phenotypic

plasticity and genetic variability between different taxa of mealybugs

• There are many morphologically cryptic taxa

which are common in many groups

• Morphological examination is time consuming and is often effective only for a particular

life stage or gender (mostly in adult females in

case of mealybugs). As a result, many cannot

be identified

• Although modern interactive versions represent a major advance, the use of keys require

high level of expertise that often lead to misidentification (Hebert et al. 2003a)

• Taxonomists have always looked for discontinuous character variations that could signal

divergence between species. The debate on

threshold values employing molecular identification for interspecific divergence is also

true in the case of morphology-based


• Early identification of new invasions is an

important aspect in preventing the spread.

Rapid and accurate identification of mealybugs is not easily accomplished with conventional taxonomy. Taxonomy separation of

many species occurring together can be difficult, particularly for the nymphal stages that

are primarily involved

Hence, there is a need for an adjunct tool that

facilitates rapid identification of species where

molecular identification, popularly called ‘DNA

barcoding’, becomes handy. The concept of

DNA barcoding was proposed by Hebert et al.

(2003b, c) as a rapid and precise way for species

discrimination of a broad range of biological

specimens using a selected 658-bp fragment of

the 5′ end of the mitochondrial cytochrome oxidase-I (mtCO-I) gene (Fig. 5.1).


Molecular Identification of Mealybugs


Fig. 5.1 (a) Organization of genes in mitochondrial

genome. (b) Arrangements of barcode region with mitochondrial membrane with barcode region (blue in colour)

and primer region (brown) with amino- and carboxyterminal spanning inside


cies whether abundant or rare, native or invasive, engendering appreciation of biodiversity,

locally and globally (Stoeckle et al. 2004).

• Opens the way for an electronic hand-held

field guide, the Life Barcoder: Barcoding links

biological identification to advancing frontiers

in DNA sequencing, miniaturization in electronics, and computerized information storage

(Stoeckle et al. 2004).

• Demonstrates value of collections: Compiling

the library of barcodes begins with the multimillion specimens in museums, herbaria, zoos

and gardens and other biological repositories

(Stoeckle et al. 2004).

• Speeds up writing the encyclopaedia of life:

Compiling a library of barcodes linked to the

vouchered specimens and their binomial

names will enhance public access to biological knowledge, helping to create an on-line

encyclopaedia of life on earth, with a webpage

for every species of plant and animal (Stoeckle

et al. 2004).

Uses of DNA Barcoding

• Works with fragments: Barcoding can identify

a species from bits and pieces. When established, barcoding will quickly identify undesirable animal or plant material in processed

foodstuffs and detect commercial products

derived from regulated species (Stoeckle et al.


• Works for all stages of life: Barcoding can

identify a species in its many forms, from eggs

and seed, through larvae and seedlings, to

adults and flowers (Rebijith et al. 2012).

• Unmasks look-alikes: Barcoding can distinguish among species that look alike, uncovering dangerous organisms masquerading as

harmless ones, and enabling a more accurate

view of biodiversity (Asokan et al. 2011).

• Reduces ambiguity: Written as a sequence of

four discrete nucleotides – CATG – along a

uniform locality on genomes, a barcode of life

provides a digital identifying feature, supplementing the more analog gradations of words,

shapes and colours (Stoeckle et al. 2004).

• Democratizes access: A standardized library

of barcodes will empower many more people

to call by name the species around them. It

will make possible the identification of spe-

The core idea of barcoding is based on the fact

that short pieces of DNA vary only to very a

minor degree within the species, and that the

variation is much less between different species.

Therefore, a threshold value of variation could be

K.B. Rebijith et al.


characterized for each taxonomic group (2–12

%), above which groups of individuals do not

belong to the same species, but form supraspecies taxon. Therefore, unknown individuals

could be assigned to a species level.


Targets for Molecular



Mitochondrial DNA

Mitochondrial (mt) DNA (Fig. 5.2) has a long

history of use at the species level; recent analyses

suggest that the use of a single gene, particularly

mitochondrial, is unlikely to yield data that are

balanced, universally acceptable, or sufficient in

taxonomic scope to recognize many species lineages (Rubinoff 2006). Mitochondrial cytochrome c oxidase subunit I (mtCO-I) gene

sequence is suitable for this role because its

mutation rate is often fast enough to distinguish

closely related species, and also because its

sequence is conserved among conspecifics and a

lack of recombination. mtCO-I sequence differences are too small to be detected between closely

related species; more than 2 % sequence divergence has been detected between such organisms, proving the barcode effective. However,

the rate of evolution of cox1 is very slow.

Fig. 5.2 Flowchart showing steps in DNA barcoding of mealybugs, from collection to sequence deposition in iBOL


Molecular Identification of Mealybugs


Advantages of Using

Mitochondrial Genome

• Haploid mode of inheritance, and it supports

less recombination

• Mitochondrial genome does not have introns

• Universal primers are robust, which can

amplify 5′ end in most of the animals, including insects

• Rapid evolution allows the discrimination of

not only closely related species but also phylographic groups within a single species

• In animal mitochondrial genome, the 13 protein coding genes are better targets because of

rare insertions and deletions (indels)

• By identifying amino acid substitution patterns of mtCO1, it is possible to assign any

undefined organisms to a higher taxonomic

group before examining nucleotide substitutions to determine its species identity



and Morphological


Mealybug specimens can be collected in 95 %

ethanol and kept at −80 °C (deep freezer) until

further work. Morphological identification can

be carried out by a taxonomist. Whenever possible, it is better to analyse at least three specimens

collected from each of the host/locality for



Genomic DNA Isolation

Total genomic DNA can be extracted from individual mealybugs using a non-destructive method

(Hajibabaei et al. 2006), while voucher specimens are required to be mounted on glass slides

and deposited with any of the National Insect

Repository such as the National Pusa Collection

(NPC) or the Indian Agricultural Research

Institute (IARI), Delhi. Various DNA isolation

protocols are available, namely (a) direct TNES

buffer method, (b) spot-PCR method, (c)


phenol:chloroform method and (d) salting out

method. Direct Buffer Method

A single insect can be crushed in 50–200 μL

YNES (50 mM Tris–HCI, pH 7.5, 0.4 M NaCI,

20 mM EDTA, 0.5 % SDS), STE (0.1 M NaCI,

10 mM Tris, pH 8.61 mM EDTA), GES (0.1 M

glycine, pH 9, 50 mM NaCI, 1 mM EDTA, 1 %

β-mercaptoethanol, 0.5 % Triton X-100) or

CTAB (100 mM Tris–HCL, pH8,1.4 M NaCI, 20

mM EDTA, 2%CTAB, 0.2 % β-mercaptoethanol)

buffer. The sample is to be incubated at 94 °C for

12 min, with the cell debris to be precipitated by

spinning it at 13,000 rpm for 1 min. The extracted

DNA is to be stored at −20 °C. Spot-PCR Method

A single insect should be crushed on a positively

charged nylon membrane soaked in a 50-mM

NaOH and 2.5-mM EDTA solution, and then

allowed to dry. A small portion (ca. 3 mm2) of the

spotted membrane is to be cut out and placed in

10–50 μL TNES, STE, GES or CTAB buffer

(described above). The sample can then be incubated at 95 °C for 10 min and cooled on ice.

Extracted DNA can be stored at −20 °C. Phenol/Chloroform Method

DNA from a single insect can be extracted using

the modification of a general procedure for

extraction with phenol (Sambrook et al. 1989;

Sambrook and Russell 2001). The insect is to be

crushed and incubated at 40 °C in 0.6 mg/mL

Proteinase K and 300 μL TNES buffer for 4–18

h. DNA can then be purified by washing with

organic solvents: once with a chloroform:isoamyl

mix (24:1 v/v); once with a chloroform:phenol

mix (1:1 v/v) and once with chloroform only.

DNA can then be precipitated with absolute ethanol. Extracted DNA can be stored at −20 °C. Salting-Out Method

DNA from a single whole insect can be extracted

using the protocol of Sunnucks and Hales (1996)

with minor adjustments, including the following:

the insect can be incubated at 40 °C in 0.6 mg/mL

K.B. Rebijith et al.


Proteinase K and TNES buffer; and the samples

can be left for at least 1 h at −20 °C during precipitation of the DNA with absolute ethanol.

Extracted DNA can be stored at −20 °C.


Polymerase Chain Reaction

Polymerase chain reaction (PCR) was developed

by Kary B. Mullis (Mullis and Faloona 1987) and

has radically changed molecular research and

diagnostics (Caterino et al. 2000). PCR involves

the in vitro synthesis of large amounts of DNA

copies from a single starting molecule and

employs short single strands of DNA (18–30

nucleotides) called oligomers or primers (Table

5.1) to select a region of specific interest from the

DNA. Once the primers are annealed to the DNA,

Taq DNA polymerase builds a complementary

strand extending from the primer by incorporating free deoxynucleoside triphosphate (dNTP:

base + deoxyribose sugar + phosphate) molecules

in the reaction mix. Two primers that anneal on

complementary strands are used, with the Taq

extending the region between them. The reaction

mixture is cycled between different temperature

optima for the different stages of reaction of

denaturation, annealing and elongation. This process is repeated in a number of cycles (usually

30–40), and the DNA thus produced increases

exponentially (Saccaggi 2006).


Sequence Analyses

and Submission

The amplified products can be eluted using an

extraction kit according to the manufacturer’s

protocol, and the sequencing can be done in an

automated sequencer (ABI prism® 3730 XL

DNA Analyzer; Applied Biosystems, USA) using

PCR specific primers, both in forward and reverse

directions. Homology search and sequence alignment can be performed employing the NCBIBLAST (http://blast.ncbi.nlm.nih.gov/) and

BioEdit version (Hall 1999), respectively.

All the sequences generated in the respective

studies need to be deposited in the NCBIGenBank and the Barcode of Life Data systems

(BOLD) (Table 5.2).


Nuclear Copies

of Mitochondrial Genes

There is a possibility that a pseudogene is being

amplified if the study encounters the following

anomalies (Zhang and Hewitt 1996):

• More than one bands, or different bands, are






• Background peaks or sequence ambiguities

are constantly found when sequencing.

• The DNA sequence contains data which will

unexpectedly change the polymerase translation of the sequence, such as unusual frameshifts, insertion/deletion or stop codons.

• The DNA sequence is particularly more divergent than expected.

• Phylogenetic analysis results in unusual,

unexplained or contradictory tree topology.

In the recent past, DNA barcoding has gained

importance in the species diagnosis of animal

species, but has some difficulty with certain

insects. This is probably due to its inconsistency

in amplifying the 5′- mtCOI region; however, a

total of 178 mtCOI sequences for 29 mealybug

species are available with the NCBI-GenBank.

Table 5.1 Primers employed in DNA barcoding of mealybugs











Amplicon Size (bp)

658 bp


Folmer et al. (1994)

649 bp

Park et al. (2010a)

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