Morphologic Character of Haematopinus Sp. Nymph and Mature

Stadium of Fries Holland Cow from Jember with SEM

(Scanning Electron Microscope)

Aan Awaludin

, Yudhi Ratna Nugraheni

, Kurniasih

, Hariadi Subagja

Department of Animal Sciences, Politeknik Negeri Jember

Department of parasitology, Faculty of Veterinary Medicine, Gadjah Mada University

Department of pathology, Faculty of Veterinary Medicine, Gadjah Mada University

Keywords: Ectoparasites, Haematopinus, Insect, Phthiriasis, SEM.

Abstract: Haematopinus is the biggest parasite of insect family of domestic animal having all lifecycles in the female,

and can only live some hours out of the female body. This research aims at seeing the ultra-structural

difference of nymph stadium with Haematopinus sp. of mature insect facing Fries Holland (FH) from Jember

district based on morphologic identification key of Meleney and Kim. Samples of nymph stadium and mature

insect of Haematopinus sp. was taken from tail end area, perineum vulvae, area, and around eyes of 5 Fries

Holland (FH) cows of each infected cow from Jember district. The samples were identified based on

morphologic identification key of Meleney and Kim and did Scanning Electron Microscope (SEM) of part of

caput and abdomen. Results of morphologic identification and ultra-structure were analyzed descriptively.

There are ultra-structural differences of nymph stadium from mature insect, especially ultra-structure of

abdomen area. Nymph stadium has not been found in gonopod development; while, in mature insect,

gonopod has developed and can be identified. SEM method can be used to change the morphologic

identification, especially in a mature insect. Nymph and mature insect of Haematopinus sp. in Fries Holland

(FH) from Jember district are Haematopinus quadripertusus species.

1 INTRODUCTION

Ectoparasites of Haematopinidae frequently parasites

in the cow. Haematopinus euryternus was reported

parasitizing on cows in areas with cold climate, while

Haematopinus quadripertusus was reported

parasitizing on cows in tropical and sub-tropical areas

(Scifield et. al., 2012).

In some cases, insect infestation was reported

causing a reduction in productivity, and some cases

were reported as a disease vector, such as babesiosis,

theileriosis, anaplasmosis and others (Norval et. al.

1984). The incidence of insect infestation in a great

number of cows causes itching, reduction in body

weight, irritating, uncomfortable feeling, reduction in

dairy water production and reduction in initial harvest

product quality (Lasisi et. al., 2010). Hadi and

Saviana (2000) suggested that incidence of insect

infestation is called as pediculosis and phthiriasis.

Haematopinus sp. has characteristics of 0.5 cm,

yellow or grey-brown with a black line of each

margin, having no eye, having three pairs of wide and

flat legs (Urquhart et. al., 1987). Haematopinus sp.

has spiracle of dorsal periphery of mesothorax (Noble

and Glenn, 1989). Size of the head is elongated with

the wider back part than front part and protrusion in

the back antenna, the antenna has 5 internodes, and

part of the thorax is wide and has sterna plate in the

lower part (Lapage, 1956).

Mouth area of Haematopinus sp. has fine and

small proboscis called as haustellum, internal part of

haustellum is completed with small teeth directing to

exit functioning to implant in female skin, having 3

prick organs whose shapes are like needles called as

stilet which may expel to function to absorb blood and

inject saliva gland into female body (Hado and

Soviana, 2000).

Meleney and Kim (1974) explained key of

identification for species of Haematopinus sp.,

namely, rounding or compact para tergite in

peripheral area of abdomen with 2 posterior setae,

posterior area of abdomen has a non-pointing

gonopod, sterna plate under thorax elongates,

Awaluddin, A., Nugraheni, Y., Kurniasih, . and Subagja, H.

Morphologic Character of Haematopinus Sp. Nymph and Mature Stadium of Fries Holland Cow from Jember with SEM (Scanning Electron Microscope).

DOI: 10.5220/0010360905690575

In Proceedings of the 3rd International Conference of Computer, Environment, Agriculture, Social Science, Health Science, Engineering and Technology (ICEST 2018), pages 569-575

ISBN: 978-989-758-496-1

569

forming an elongating head and having parasite in

cows, so that the species directs to Haematopinus

euryternus species or maybe Haematopinus

quadripertusus. Female Haematopinus euryternus

species has body with 2.23 – 3.18 mm in long and

male Haematopinus euryternus species has body with

1.99 – 2.7 mm in long, having short and round

processus anterolatera; sterna plate, thin abdominal

trachea, subgenital plate median forming 9

subtrapezoid, tergite in abdomen with elongating and

more protruding processus anteromedial, short

forehead, and having subgenital plate in male insect,

completed with 6 anterior setae. Female

Haematopinus quadripertusus species has 3.42 –

4.75-mm body and male Haematopinus

quadripertusus species has 3.04 – 3.52-mm body,

having elongate processus anterolateral sterna plate,

long forehead, elongate processus anterolateral in

sterna plate thorax in female insect, thick abdominal

trachea trunks, long and narrow gonopod, wide

subgenital plate median subrectangular, 9

abdominal tergite has short and blunt processus

anteromedial, and male insect has subgenital plate

completed with 4 anterior setae.

Lapage (1956) explained lifecycle of

Haematopinus sp., 1-mm Haematopinus egg,

elongating shape, white in color, and sometimes

brown, eggshell is not hard, female insect lays one or

four eggs per day and female insect can expel 24 eggs,

eggs will hatch to be nymph (young insect) after 9 –

19 days at 27.5

C, nymph will come out from egg

and grow and molt perfectly and grow into adult.

2 METHODOLOGY

2.1 Location of Research

Samples of nymph and mature stadium of

Haematopinus sp. Were taken from Fries Holland

(FH) cows found suffering from pediculosis

(ptiriasis_ from Jember district.

The research was conducted in some locations.

Morphologic identification of insect was conducted

in the Parasitological laboratory, Faculty of

Veterinary Medicine, Gadjah Mada University,

Yogyakarta. Length scale and picture of insect were

measured using Lucida camera in Plant Infest

laboratory (Nematological Laboratory), faculty of

farming, Gadjah Mada University, Yogyakarta. ultra-

structure was identified by Scanning Electron

Microscope (SEM) in Central Research of Zoology,

Institute of Sciences of Indonesia (LIPI), Cibinong.

2.2 Tools and Materials

Tools to take samples included rubber glove, 2-ml

microtube, pinset, and labeling paper. Tools to

identify insect morphology included petri dish, slide

glass, cover glass, measurer (0.05-mm accuracy),

Lucida camera, pencil, HVS paper and tracing paper,

tools to examine body surface ultra-structure included

a holder, stibe, vacuum evaporator, Ion Coates, and

JEOL JSM-5310LV Scanning Electron Microscope

(SEM).

Required materials were absolute ethanol, 50%

ethanol, 70% ethanol, 85% ethanol, 95% ethanol,

2.5% glutaraldehyde, coccodylate buffer, 2% tannin

acid, 1% osmium tetraoxide, tertiary butanol, and

aquades.

2.3 Methods

2.3.1 Collection of Samples

Samples of nymph and mature stadium of

Haematopinus sp. In Fries Holland (FH) found

suffering from pediculosis (phthiriasis) were taken

from the tail area, especially fiber tail end, around

perineum vulvae, ear, and surrounding eyes of the

cow with 5 mature and nymph insects, using pinset,

then inserted into 2-ml microtube containing absolute

ethanol and labeled. Samples of mature insects and

nymph were collected in a separate microtube.

2.3.2 Morphologic Identification

Morphologic identification of mature insects and

nymph samples included macroscopic and

microscopic observations.

Macroscopic observation consisted of

identification of body, color, presence or absence of

wings, total extremities, and long measures body

using measurer.

Microscopic observation consisted of

morphological observation of caput, thorax,

abdomen, extremity, and picture and measure scale

were made using a binocular microscope (zooming 4

x 10) completed with Lucida camera drawing the

shadow of sample objects using pencil on HVS paper

which was then moved to tracing paper.

2.3.3 Identification of Ultra-structure

Identification of ultra-structure with Scanning

Electron Microscope (SEM) included caput and

abdomen. Observation of ultra-structure in caput part

included an anterior end to see ultra-structure of

mouth type. Observation of ultra-structure in

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abdomen part included pleural disk part to see ultra-

structure of para tergite and posterior setae and in the

posterior end, part to observe ultra-structure of

gonopod and subgenital plate median.

The process of Scanning Electron Microscope

(SEM) was conducted in Central Research of

Zoology, Institute of Sciences of Indonesia (LIPI),

Cibinong. Before the samples were delivered, the

samples were fixed in absolute ethanol.

The process of Scanning Electron Microscope

(SEM) included process of specimen preparation

consisting of 5 stages: first, the samples were washed

by soaking in coccodylate buffer (2 hours), then the

samples were agitated in Ultrasonic cleaner (5

minutes); second, the samples were prefixed by

inserting the samples in 2.5% glutaraldehyde (2-3

hours); third, the samples were fixed by using 2%

tannin acid (6 hours), then washed by coccodylate

buffer (5 minutes), the process of washing was

conducted repeatedly until 4 times, then processed by

second washing by 1% osmium tetraoxide (one hour),

third washing by aquades (15 minutes); fourth,

dehydration process was conducted by soaking the

samples in stratified ethanol of 5% ethanol ( 5

minutes), this process was continued by using 70%

ethanol (20 minutes), 85% (20 minutes), and 95% (20

minutes) and absolute ethanol (10 minutes)

conducted two times at room temperature; fifth,

drying process by soaking the samples in tertiary

butanol (10 minutes), this process was conducted

until 2 times, then frozen by Freezed Drier until dry.

The process of Scanning Electron Microscope

(SEM), the samples which had been covered by

copper with Ion Coates (15 minutes) and observed by

Scanning Electron Microscope (SEM) of JEOL JSM-

5310LV.

3 RESULTS AND DISCUSSION

Fries Holland (FH) from Jember found suffering

from pediculosis (phthiriasis) had nymph stadium and

mature insect of Haematopinidae family. The

ectoparasites infestation area covered areas of the

hairy tail end, around the ear, and around perineum

vulvae, especially in the lower part. In the area, insect

eggs binding to hair were also found.

3.1 Morphologic Identification

Nymph stadium of Haematopinus sp. In Fries

Holland (FH) from Jember was found black, body

divided into 3 parts (caput, thorax, and abdomen),

dorso-ventral flat body shape, having no wings,

having 3 pairs of legs (proleg, mesoleg, and metaleg)

where each leg is divided into 4 internodes (coxa,

femur, tibia, tarsus) with 1 claw in each leg (Figure

1). The mature insect of Haematopinus sp. In Fries

Holland (FH) from Jember was black-grey in color, a

body is divided into 3 parts (caput, thorax, and

abdomen), dorso-ventral flat body shape. Having no

wings, having 3 pairs of legs

(proleg, mesoleg, and

metaleg) where each leg is divided into 4 internodes

(coxa, femur, tibia, tarsus) with 1 claw in each leg

(Figure 2). Body lengths of nymph samples of

Haematopinus sp. In Fries Holland (FH) from

Jember are 3.10 mm, 2.60 mm, 2.90 mm, 2.75 mm,

and 2.95 mm (Table 1), or averagely body length of

nymph samples is 2.86 mm. Body lengths of mature

insect samples of Haematopinus sp. In Fries Holland

(FH) from Jember are 4.70 mm, 4.60 mm, 4.20 mm,

4.15 mm, and 4.20 mm (Table 2), or averagely body

length of mature insect is 4.37 mm.

Figure 1. Nymph of Haematopinus sp. Of Fries Holland

(FH) from Jember: I. Ventral ( a. antenna, b. claw, c. tarsus,

d. tibia, e. femur, f. coxa, g. sterna plate, h. abdominal

tracheae trunks, i. gonopod); II. Dorsal (A. caput, B. thorax,

C. abdomen, i. para tergite, j. setae). Scale bar: 0.222 mm.

Figure 2. The mature insect of Haematopinus sp. Of Fries

Holland (FH) from JemberL I. Ventral (a. antenna, b. claw,

c. tarsus, d. tibia, e. femur, f. coxa, g. sterna plate, h.

Morphologic Character of Haematopinus Sp. Nymph and Mature Stadium of Fries Holland Cow from Jember with SEM (Scanning Electron

Microscope)

571

abdominal tracheae trunks, i. gonopod); II. Dorsal (A.

Caput, B, Thorax, C. Abdomen, j. para tergite, k. setae).

Scale Bar: 0.222.

Table 1. Samples of the nymph of Fries Holland (FH) from

Jember.

Identification Result

Color : Black

Body Shape : Dorsoventral flat

Body parts : 3 parts (caput,

thorax, abdomen)

Caput

 Shape : Narrow and

pointing (smaller

than thorax part )

 Mouth type : Pricking and

absorbing

 Total antennas : 2 pairs

 Antenna internodes : 5 internodes

 Eye : Absent

Thorax

 Shape : No segment

 Total extremities : 3 pairs: Proleg,

Mesoleg, Metaleg

 Extremity internodes : 4 ruas: Coxae,

Femur, Tibia,

Tarsus

 Claw : Single

 Wing : No wing

Abdomen

 Shape : Segmental

 Paratergite : Rounding, having

pleural disks with

2 pairs of setae in

angular part

 Respiratory tract : Abdominal

tracheae tract is

seen clearly

 Reproduction tool : Unobservable

Body length size

 Nymph 1 : 3.10 mm

 Nymph 2 : 2.60 mm

 Nymph 3 : 2.90 mm

 Nymph 4 : 2.75 mm

 Nymph 5 : 2.95 mm

Table 2. Samples of mature insect of Fries Holland (FH)

from Jember

Identification Result

Color : Black-grey

Body Shape : Dorsoventral flat

Body parts : 3 parts (caput,

thorax, abdomen)

Caput

 Shape : Narrow and

pointing (smaller

than thorax part)

 Mouth type : Pricking and

absorbing

 Total Antenna : 2 pairs

 Internodes : 5 internodes

 Eye : Absent

Thorax

 Shape : No segment

 Total extremities : 3 pairs: Proleg,

Mesoleg, Metaleg

 Internodes : 4: Coxae, Femur,

Tibia, Tarsus

 Claw : Single

 Wing : No wing

Abdomen

 Shape : Segmental

 Paratergite : Rounding, having

5 setae hairs in

angular part

 Respiratory tract : Abdominal

tracheae tract is

seen clearly

 Reproduction tool : Long and narrow

Gonopod

Body length size

 Insect 1 : 4.70 mm

 Insect 2 : 4.60 mm

 Insect 3 : 4.20 mm

 Insect 4 : 4.15 mm

 Insect 5 : 4.20 mm

Results of observation for nymph samples and

mature insect) Table 1 and Table 2) macroscopically

and microscopically indicate that there is no

morphologic difference of nymph from mature insect,

except nymph samples are seen black than the mature

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insect. Key of morphologic identification of

Haematopinus sp., according to Meleney and Kim

(1974) is that abdomen has round and compact para

tergite with 2 posterior setae, and gonopod is not

pointing in posterior part. Haematopinus

quadripertusus has body lengths of 3.42 – 4.75 mm

(female) and 3.04 – 3.52 mm (male), having thick

abdominal tracheae trunks, long and narrow gonopod.

Haematopinus euryternus species has body length

sizes of 2.23 – 3.18 mm in female and 1.99 – 2.7 mm

in male, thin abdominal trachea, short and compact

gonopod.

Based on the key of identification, samples of

insect are from Haematopinus quadripertusus species.

3.2 Identification of Ultra-structure

Ultra-structure of the anterior end of nymph samples

and mature insect (Figure 3 and Figure 4) is seen as

pointing shape of the suction tube in haustellum with

similar function to proboscis, labrum (lip), and 4 pairs

of setae. Organ of suction tube in haustellum belongs

to an insect with suction tube functioning to suck

blood from hospes as main food insect and flexible to

pull and expel according to need. Haustellum is an

organ with similar function to mouth of higher level

animals. Whitlock (1960) explained that

Haematopinus sp. Is different from other insects with

blood suction tubes because they have no long

proboscis pricked into the female body. Hadi and

Soviana (2000) indicated that Haematopinus sp. Has

mouth consisting of the fine and small proboscis

(haustellum). The internal part of haustellum is

completed with small teeth directing to exit to implant

into the female skin, having 3 organs of the suction

tube to suck blood and inject saliva gland into a female

body.

Figure 3. Ultra-structure of the ventral anterior end of

Haematopinus sp. Nymph of Fries Holland (FH) from

Jember: a. suction tube, b. Labrum, c. Setae. Scale bar:

58.00 m.

Figure 4. Ultra-structure of the ventral anterior end of

mature insect of Haematopinus sp. Of Fries Holland (FH)

from Jember: a. suction tube, b. Labrum, c. Setae. Scale

Bar: 58.00 m.

Ultra-structure of abdomen area of pleural disk

part of nymph samples and mature insect of

Haematopinus sp. (Figure 5 and Figure 6) indicates

that para tergite is rounded with spiraculum in end

part and has 2 setae hairs in posterior part in the lateral

area of the abdomen. In ventral part, mature female

insect samples have elongated and narrowing

gonopod shape and posterior part of gonopod has

subgenital plate median in subrectangular shape with

hollow in the central part (Figure 8), different from

the picture of ultra-structure in nymph samples

unseen for gonopod shapes (Figure 7). According to

Meleney and Kim (1974), Haematopinus

quadripertusus has long and narrow gonopod and

subgenital plate median of subrectangular shape.

Figure 5. Ultra-structure of pleural (ventral) disk of

Haematopinus sp. Nymph of Fries Holland (FH) from

Jember: a. spiraculum, b. Setae. Scale bar: 101.54 m.

Morphologic Character of Haematopinus Sp. Nymph and Mature Stadium of Fries Holland Cow from Jember with SEM (Scanning Electron

Microscope)

573

Figure 6. Ultra-structure of pleural (ventral) disk of

Haematopinus sp. A mature insect of Fries Holland (FH)

from Jember: a. spiraculum, b. Setae. Scale bar: 101.54 m.

Figure 7. Ultra-structure of ventro posterior nymph of

Haematopinus sp. Of Fries Holland (FH) from Jember

Gonopod shape is not seen (arrow). Scale bar: 135.38 m.

Figure 8. Ultra-structure of ventro posterior mature insect

of Haematopinus sp. Of Fries Holland (FH) from Jember

Gonopod shape is not seen (arrow). Scale bar: 135.38 m.

Based on identification key of Meleny and Kim

(1974), specially strengthened by the picture of ultra-

structure of mature insect sample consisting of a

suction tube, para tergite rounding with 2 setae hairs

in posterior part, long and narrow gonopod,

subrectangular subgenital plate median, then the

sample was identified as Haematopinus

quadripertusus species. Identification of insect using

Scanning Electron Microscope (SEM) can be used to

strengthen morphologic identification of insect,

especially in the mature stadium. In nymph,

especially in the posterior end, it cannot be a

reference of identification because gonopod did not

have developed, but the shape of pleural disk part

could be observed.

4 CONCLUSIONS AND

RECOMMENDATIONS

Scanning Electron Microscope (SEM) method can be

used to strengthen morphologic identification of

insect of Haematopinus sp. In mature stadium.

Samples of nymph and mature insect of Fries

Holland (FH) from Jember is Haematopinus

quadripertusus species.

Recommendations

Research of identification of species needs to add

molecular methods to see the phylogenetic tree of

researched species.

ACKNOWLEDGEMENT

Researcher team thanks, Prof. drh. Kurniasih,

M.VSc., Ph.D. for guidance and fund have given for

individual research so that this research could be

conducted. The team also thanks, Dr. drh. R. Wisnu

Nurcahyo for all guidance forms and

recommendations, parasitological department of

Veterinary Medicine Faculty and Laboratory of Plant

Infest (Nematology Laboratory), Faculty of Farming,

Gadjah Mada University, Yogyakarta, who had

provided place and tools for these research activities.

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