Detection and identification of hidden infestation by Callosobruchus species belonging

to family bruchidae
Subham kumar1, Sumit kumar2, Kavita Gupta3
1,2
Department of Plant Protection, C.C.S. University Campus, Meerut -250004
3
Division of Plant Quarantine, NBPGR, PUSA Campus, New Delhi – 110012
Abstract: The present study, three species viz. C. chinensis, C. maculatus and C. analis were taken up
for studying their detection, identification and management. Since these insects cause hidden infestation,
the detection was done through X-ray radiography using MX 200 Faxitron machine which is for real-time
detection of insects inside the grains. After detection, the insects were retrieved and mounted for their
identification up to species level. The identification was undertaken based on morphological characters of
the three species. The differentiating characters including those of antenna, elytra, hind leg, pygidium and
genitalia were studied for all the three species. Management options available such as mechanical cleaning,
salvaging through X-ray, fumigation using EDCT (Ethylene Dichloride Carbon Tetrachloride) mixture at
normal atmospheric pressure (NAP) and ambient temperature and vacuum fumigation have also been
discussed.
Keywords: X-ray radiography machine, infested seed samples (mungbean, cowpea, gram and pigeon
pea), Electron microscope, fumigants (EDTC) and fumigation chamber (Atmospheric and Vacuum
fumigation).
Introduction: The members of the family Bruchidae have long been reported to destroy the seed of the
leguminous plants but a number of them are now known to attack the seed or are associated with the
flowers or leaves of plants belonging to other families such as Compositae, Malvaceae, Convolvulaceae,
Anacardiaceae, Rosaceae, Umbelliferae, Papaveraceae and Palmae. They are not only serious pests of the
edible pulses, beans, lentils and peas but also attack the pods and the seeds of the wild forest, medicinal and
ornamental plants. The available literature on bruchidae shows that over 1,500 species have so far been
recorded from different parts of the world. The bruchidae fauna of India, however, has not been properly
explored and very few species are represented in the collections of the Indian Agricultural Research
Institute, New Delhi and Forest Research Institute, Dehradun, and even these are neither properly identified
nor adequately described. At NBPGR, the world bruchid literature was compiled and a database on
bruchids developed. It also included digitized keys for their identification Research work has accordingly
been carried out and collections have been made of bruchids from different parts of North-West India with
a view to study and establish the identity of the various species, listing their host plants, determining their
specific ranges and assigning them to their appropriate systematic positions. The present study was
undertaken to detect the presence of bruchid infestation in mungbean, cowpea and gram seeds through X-
ray image analysis and to identify the retrieved.

Materials and Methods:

The present investigation on “Detection and identification of hidden infestation by Callosobruchus
species belonging to family bruchidae” were carried out in the Entomology laboratory, division of plant
quarantine, at NBPGR, New Delhi during 2012-2013.
Infested seed materials: Four infested seed samples namely: mungbean, cowpea, gram and pigeon pea
were collected from the division of germplasm conservation, NBPGR, New Delhi. The seed sample were
brought to the Entomology laboratory of division of plant quarantine at NBPGR, New Delhi for further
study.
 Mechanical cleaning: All the seed lots were cleaned mechanically (hand picking) by removing
the insects, stages thereof, infested, discoloured, deformed and shriveled seeds.
Grain material for standardization of X-ray radiography: Experiments for standardization of
radiography were undertaken on mungbean, cowpea, gram and pigeon pea.
X-ray machine: The X-ray machine available at National Bureau of Plant Genetic Resources is standard
cabinet X-ray machine of Faxitron series. The machine gives soft X-ray radiation enough for studying seed
in smaller quantities. The machine has all built in safety features protecting those working in the vicinity
from exposure to the radiation (Fig.1).

Fig.1. Faxitron SR MX 200 real-time X-ray machine for detection of hidden infestation of
bruchids in seeds
The seeds are spread in a small plastic tray and exposed to soft X-rays generated at 19-26 kv. For
19-25 second depending on the seed size using the real time X-ray machine, Model No.
Faxitron SR MX-200. As the machine is real-time, it automatically gives the X-ray
radiograph on the monitor (Fig.2).

Fig.2. Image of seed plate after X-ray exposure.

Infested seeds are clearly distinguishable from healthy ones on the monitor. The exposed plate is
gently removed from the X-ray machine without disturbing the seed geometry. The infested seeds
(as seen in the X-ray radiograph) are then handpicked from the seed sample with original geometry
retained an X-ray tray/ plate.
Identification of interceptions: The infested seeds separated by handpicking after X ray radiography
are soaked overnight, the seed teased open and the insect removed from inside (Fig.3).
 (a). Mung bean (b). Cow pea

(c). Gram (d). Pigeon pea

Fig.3. Infected seed collected through X-ray Radiography machine and after soak in water

Examined the insect stage by binocular microscope to identify the taxonomic and morphological
characters with the help of identification key (Fig.4).

Fig.4. Examined the insect stage by binocular microscope

RESULTS AND DISCUSSION:
The following section describe in details the various insect pests detected with the help of x-ray
Radiography machine. Their taxonomic details are also elaborated and the morphological characters, life
cycle as study have been described:
Test insects:
i. Callosobruchus chinensis
ii. Callosobruchus maculatus
iii. Callosobruchus analis
1. Taxonomic characters of Callosobruchus chinensis
Head: Brown or black broader at posterior and frons craniate, its surface covered with white setae. Eyes
bulbous, strongly emarginated and surface of canthus covered with white or golden setae, maxillary palpi
black, protruding. Antennae dark brown, segments 3-11 serrate in female, pectinate and often margined
with black in male.
Pronotum: Sub-conical, dark brown or black, its surface covered with light brown setae, with two medium
prominent callosities in its posterior region, callosities covered with shining white and orange setae.
Scutellum: Squarish and covered with white setae.
Elytra: Elongate, quadrangular, rounded at apical margins, their ground colour entirely reddish or red
intercepted by black spaces, striae punctuate, striae 3-8 abbreviated at apex, a pair of tubercles present at
the bases of 3rd and 4th striae, humeral callosities well developed, surface of each elytron covered with three
transverse rows of setae, a broad row of golden setae in the anterior region and two narrow rows of white
setae in the middle, the rest of the elytral surface not covered with setae.
Legs: Usually reddish with the pretarsi black, sometimes dark brown with the coxa and hind femora black,
bicarinate on inferior margins, each carina bearing an apical tooth, outer tooth larger and blunt, inner tooth
narrow and pointed, hind tibia produced into a spine at its internal apical end.
Pygidium: Dark brown or black, vertical, uniformly covered with pale setae in male, oblique and with
distinct median row of white setae and scarce pale setae elsewhere in female (Fig. 5).

(a). (b).

(c). (d)
 Fig.5. Callosobruchus chinensis identified in infested mungbean seed through stereobinocular
Microscopic observations (a). Male, (b). Female, (c). Male pygidium, and (d). Female pygidium

Phallus: Narrow, elongated, 1.73 mm long, parameres flattened at their distal ends, each provided with a
distinct apical papilla carrying a pair of setae, success devoid of setae, with a pair of lateral chitinized
toothed plates, exophallic valve acuminate, proximal lobe of the spermatheca broad, distal lobe short and
narrow.
Size: Small sized 3.23 to 3.36 mm in male and 3.43 to 3.56 mm in female.
Hosts: Arachishypogoea, Cajanuscajan, C. indicus, Cassia javanica, C. tora, Cicer arietinum, Cyamopsis
tetragonolobus, Dolichus biflorus, D. lablab, D. lablab var. lignosus, Dolichus monocalis, Dolichus sp.,
Glycine hispida, Lathyrus odoratus, L. sativus, Lens esculenta, Lens sp., Nelumbo nucifera, Phaseolus
aconitifolius, P. angularis, P. articulatus, P. aureus, P. calcaratus, P. lunatus, P. Mungo, P. radiatus, P.
vulgaris, Pisumarvense, P. sativum, Viciafaba, Viciafaba var. equina, Viciasp., Vignacatjang, V. catjang
var. sinensis, V. cylindrica, V. oligosperma, V. sesquipedalis, V. sinensis, V. unguiculata,
Voandzeiasubterranea.
Distribution: Widely distributed in plains and hills all over India.
2. Taxonomic characters of Callosobruchus maculatus (F.)
Head: Black, elongate, frons carinate, eyes emarginated, canthus broad and covered with pale setae.
Antennae testaceous, dark brown or black, subserrate in female, longer and serrate in male.
Pronotum:Subconical, usually black, sometimes dark brown and with a pair of posterior median callosities
covered with dense pale setae on the rest of the surface thinly scattered with a pair of prominent lateral
white spots in female.
Scutellum: Quadrangular and bearing white setae.
Elytra: Elongate, about twice as long as broad, with 4 th and 5thstriae stopping short of other striae; their
ground colour in the female commonly testaceous having well defined median and apical black and
occasionally with an additional humeral black spot but may be entirely testaceous or entirely black, white
pubescence on each elytron disposed along the median black spot but may be entirely testaceous or entirely
black, white pubescence on each elytron disposed along the median black spot as a longitudinal band of
white setae on the third interstria making the mesal border, a narrow transverse band on the 4 th -
9thintersteriae forming the anterior edge, and a broader band across the same interstriae comprising the
posterior edge, the white pubescence thus assuming the form of two C-shaped areas with their bases facing
each other; the ground colour of elytra in the male usually testaceous with median or apical black naked
areas or both sometimes entirely testaceous, the testaceous parts covered over by golden setae.
Legs: Testaceous, hind legs sometimes dark brown, hind femur bicarinate below, inner carina with a
narrow pointed tooth near apex, outer carina with a short comparatively blunt process.
Pygidium: In the female oblique, either entirely testaceous or black interrupted by a median testaceous
stripe, the latter covered over by white pubescence which may spread laterally along the anterior border for
varying extent, rest of the surface carrying sparse golden setae (Fig. 13d), pygidium in the male vertical,
either entirely testaceous or testaceous with black posterolateral spots, its surface uniformly clothed with
golden setae which may aggregate densely to from median line (Fig.6).
 (a) . (b).

(c). (d).

Fig.6. Callosobruchus maculatus identified in infested cowpea seed through stereobinocular

microscopic observation (a). Male, (b). Female, (c). Male pygidium, and (d). Female pygidium

Phallus: short, broad, 1.57 mm long, parameres elongated, free and flattened at tips which reach beyond
the tip of exophallic valve, success region of endophallus beset with numerous thickly set lateral spines, its
two lobes of the spermatheca almost sub-equal and the proximal slightly broader than the distal.
Size: moderate, length 3.29 mm to 4.95 mm in male and 4.01 mm to 4.81 mm in female.
Host:Acacia arabica, Arachis hypogea, Cajanuscajan, C. indicus, Cercis Canadensis, Cicerarietinum,
Dolichusbiflorus, D. Cyprus, D. lablab, D. monocalis, D. sesquipedalis, D. sudanensis, Dolichussp.,
Glycine hispida, G. max, Gossypiumsp.,Lathyrusclymenum, L. sativus, Lens esculenta, Medicagociliaris,
Phaseolusaconitifolius, P. acutifolius, P. angularis, P. articulatus, P. aureus, P. Lunatus, p. mungo, P.
radiatus, P. vulgaris, Pisumsativum, Pisumsp.,Viciaervilia, V. faba, Vignacatjang, V.unguiculata,
Voandzeiasubterranea.
Distribution: Widely distributed in plains and hills all over India.
3. Taxonomic characters of Callosobruchus analis (F.)
Head: Small, dark brown, frons carinate, covered with pale setae, antennae testaceous, sub-serrate, eyes
emarginated, canthus broad and clothed with pale white setae.
Pronotum: Sub-conical, dark-brown, surface covered with sparsely scattered golden setae, a prominent
double patch of whitish setae present at its posterior end in the middle and a median longitudinal depressed
line in line with the frontal carina.
Scutellum: Quadrangular, covered with dull white or shining white setae.
Elytra: Dark-brown or light brown, each with dull central and apical black spot, the two joining each other
along their outer ends, each elytra about twice as long as broad, with striae 4 and 5 stopping short of the
other striae near the apical end, humeral callosities well developed, brown parts of the elytra covered over
with golden setae, black spots naked, a prominent oval patch of white setae present in between the black
spots and third interstria sometimes covered by white setae in front of the white patches (Fig. 16a and b).
Legs: Testaceous, hind pair darker than the others, hind femur bicarinate below, each carina with a sub-
apical spine, inner spine short and pointed, outer spine larger and blunt and hind tibia flattened and
produced at its apical outer end into a prominent process.
Pygidium: Black, vertical in male, oblique in female, with a median band of white setae in both the sexes
(Fig.7).

(a). (b).

(c). (d).
Fig.7. Callosobruchus analis identified in infested gram seed through stereobinocular
microscopic observations. (a). Male, (b). Female, (c). Male pygidium, and (d). Female pygidium

Phallus: Moderately developed, 1.41 mm long, parameres completely free, flattened at ends into spoon-
like structure, saccus of endophallus with a few thick pointed medium spines and a pair of lateral
chitinizedtoothed plates, rest of the endophallic surface covered with short blunt tubercles; exophallic
valve larger, conical; proximal lobe of spermatheca elongated and broad, distal lobe short and pointed.
 Size: Moderate, length 3.64 mm to 3.96 mm in male and 3.22 mm to 3.89 mm in female.
Host: Abrus precatorius, Acacia arabica, Cajanus cajan, C. indicus, Canavalia ensiformis, Cicer
arietinum, Crotolaria sp., Dolichos biflorus, D. cyprus, D. lablab, D. lablab var. lignosus, D. lablab var.
typicus, Glycine hispida, G. max, Lathyrus sativus, Lens esculenta, Phaseolus acontifolius, P.
acutifolius, P. articulatus, P. aureus, P. calcaratus, P. lunatus, P. mungo, P. radiatus, P.
vulgaris,Physostigma venenosum, Pisum sativum, Vicia faba, Vigna catjang, V. sesquipedalis, V.
sinensis, V. unguiculata and Voandzeia subterranea.
Distribution: Widely distributed in plains and hills all over India.
Life cycle of bruchid: Adult beetle is 3-4 mm long, female being larger, brownish in colour, broader at
shoulders and rounded posteriorly. There are dark patches on elytra and thorax. Adults show sexual
dimorphism. Males possess deeply emarginated or indented eyes and prominently serrate antennae, while in
female these characters are not distinctly marked. Tip of abdomen in females is exposed while in males it is
covered by elytra. They are active beetles and readily fly when disturbed. Fecundity is about 100 eggs per
female. Eggs are whitish, elongated and stuck on the grains or on pods and sometimes on the surface of the
container. Incubation period is 3-6 days. Grubs are scarabeiform or cruciform, plump and with short legs and
yellowish in colour. First instar larvae bear functional legs and a pair of thoracic plates to facilitate boring
into the seeds. They feed on the inner contents of the grain and may damage several grains during
development. Larval period may vary between 12 and 20 days. Pupation takes place inside the grain and
pupa is dark brown in colour. Occasionally, pupation may take place outside the grain in a cocoon made of
excretory matter. Completion of life cycle takes 4-5 weeks and there may be 6-7 overlapping generations in
a year (Fig. 8).

Fig.8. Life cycle of bruchid

Disinfestations treatments in quarantine: Disinfestations treatment commonly used in quarantine
include fumigation, X-ray radiography, mechanical cleaning, etc. Apart from these, irradiation,
cold, heat, controlled atmosphere and combination treatments are gaining popularity as these are
supposedly safe and environment friendly. Their use is being extensively explored world-wide
as alternatives to the use of chemicals, especially the most widely used quarantine fumigant,
methyl bromide and Ethylene Dichloride- Carbon Tetrachloride, which has been designated as an
ozone layer depleting substance and is likely to be phased out in near future.
 Table 1. Showing sexually dimorphic characters in Callosobruchusspp.

S. No. Name of species Male Female

1. C. chinensis (L.) Antennae long and pectinate; a pair Antennae short and sub-
of tubercles present at the bases serrate; elytral tubercles
of 3 r d a n d 4 t h s t r i a e o f each absent.
elytron.

2. C. maculatus (F.) Antennae long and deeply serrate;
elytra without distinct C-shaped
areas of white pubescence;
pygidium uniformly covered
with golden setae.
Antennae short and sub-
serrate; white pubescence
on the elytra forming two
distinct C-shaped areas
with their bases facing
each other; pygidium
with a pair of black
postero-lateral spots.

3. C. analis (F.) Each elytron with a pair o f b l a c k Each elytron with a pair of
n a k e d s p o t s separated by a dark black spots separated by
patch of dull white setae; a patch of shining white
Pygidium vertical. Antennae setae; pygidium oblique.
slightly thick. Antennae slightly thin.
Summary:
The genus Callosobruchus has been of concern as the major pests of legumes in field
as well as in storage. Its species are destructive to leguminous seeds and some of the major
hosts of Callosobruchus species are Cicer, Cajanus, Cyamopsis, Cynodon, Dolichos, Glycine,
Lens, Pisum, Phaseolus, Pueraria, Sesbania, Vicia, Vigna and Voandzeia. The genus
Callosobruchus Pic. is comprised of about 30 species, distributed in warm parts of the Old
World, a few of which are introduced to the new world; of these, 19 species have been
reported from various geographical regions of India viz. Callosobruchus analis, C.
antennatus, C. cajanus, C. chinensis, C. dolichosi, C. indica, C. maculatus, C. madurensis, C.
montanus, C. nigripennis, C. orientalis, C. phaseoli, C. theobromae, C. anjaliae, C.
sukhanensis, C. multinigrus, C. antenniferus, C. sasangirensis, and C. stoutus. However, in
all of above mentioned species, some of the major pest species are C. chinensis, C.
maculatus, C. analis, C. cajanus, C. phaseoli, and C. theobromae, which caused major
damage to pulses during the storage.

Therefore, during the present study, three species viz. C. chinensis, C. maculatus and
C. analis were taken up for studying their detection, identification and management. Since
these insects cause hidden infestation, the detection was done through X-ray radiography
using MX 200 Faxitron machine which is for real-time detection of insects inside the grains.
After detection, the insects were retrieved and mounted for their identification upto species
level. The identification was undertaken based on morphological characters of the four
species. The differentiating characters including those of antenna, elytra, hind leg, pygidium
and genitalia were studied for all the three species. Management options available such as
mechanical cleaning, salvaging through X-ray, fumigation using EDCT (Ethylene Dichloride
Carbon Tetrachloride) mixture at normal atmospheric pressure (NAP) and ambient
temperature and vacuum fumigation have also been discussed. The present study gives an
insight into the detection, identification and management aspects of the Callosobruchus spp.
and these findings would be a useful reference for future students and researchers interested
in the important field of storage entomology.

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Arora, G.L. (1977). Taxonomy of the bruchidae (Coleoptera) of northwest India. Part I.
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Bhalla, S., Kapur, M.L., Lal, B., Verma, B.R. and Singh, C. (2002). Quarantine risk
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Gupta, K., Ruquaeya, B., Gupta, C.S., Bhalla, S., Kapur, M.L., Lal, B. and Singh, C. (2011).
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Pajni, H.R. and Gupta, I.J. (1975). Callosobruchusindica sp. nov.from Chandigarh
(Coleoptera: Bruchidae). Orient. Insect.,9: 447-449.

Singh, S.C. and Kumari, R. (2000). A study of the biology of Callosobruchuschinensis (L.)
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Singh, T. (1979). A key to the north-west Indian Bruchidae. Entomologist’s Monthly

Magazine,113: 219-231.
Singal, S.K. and Pajni, H.R. (1990). Six new species of Callosobruchus Pic.from India
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