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Differential Haemocyte Counts (DHC) Differential Haemocyte Counts (DHC) in the IVin the IVthth Instar Larvae of Red Instar Larvae of Red Palm Weevil, Palm Weevil, Rhynchophorus Rhynchophorus

ferruginusferruginus

Mohammed S. Al-Khalifa and

M. I. SiddiquiZoology Department, College of Science,

King Saud University,P. O. Box 2455, Riyadh 11451

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بسم الله بسم الله الرحمن الرحيمالرحمن الرحيم

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IntroductionIntroduction

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Morphologically, the haemocytes of Arthropods Morphologically, the haemocytes of Arthropods and other invertbrates are comparable to the and other invertbrates are comparable to the

vertebrate leucocytes (Jones, 1962, 1975, vertebrate leucocytes (Jones, 1962, 1975, Wigglsworth 1979, Gupta 1979, 1990, 1991, Han Wigglsworth 1979, Gupta 1979, 1990, 1991, Han

and Gupta 1989 and Bardoloi and Hazarika and Gupta 1989 and Bardoloi and Hazarika 1992, Al-Khalifa, 2003) constitute an important 1992, Al-Khalifa, 2003) constitute an important

part of the haemolymph of part of the haemolymph of Rhynchophorus Rhynchophorus ferrugineusferrugineus . .

These cells were categorized into different These cells were categorized into different

types by Millara 1947. types by Millara 1947.

Plasmatocytes and Granlar haemocytes earned Plasmatocytes and Granlar haemocytes earned the name of immunocytes on the basis of their the name of immunocytes on the basis of their

phagocytic ability.phagocytic ability.

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Informations on the haemocytes of Informations on the haemocytes of R. R. ferrugineusferrugineus are meager and due to its are meager and due to its serious economic impact on the date serious economic impact on the date

palm as a destructive pest, it has been palm as a destructive pest, it has been chosen for this study.chosen for this study.

Differential Haemocytes Counts are the Differential Haemocytes Counts are the

indicator of the exposure to the indicator of the exposure to the insecticides or its pathological insecticides or its pathological

conditions. Hence present study has conditions. Hence present study has been carried out.been carried out.

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Materials and MethodsMaterials and Methods

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• Colony of the red palm weevil Colony of the red palm weevil R. ferrugineusR. ferrugineus was was established in the laboratory, Zoology established in the laboratory, Zoology Department, King Saud University to supply the Department, King Saud University to supply the grubs of the known stage larvae.grubs of the known stage larvae.

• The colony was maintained at 28± 2ºC and 75% The colony was maintained at 28± 2ºC and 75%

relative humid\ity and fed on the sugarcanes relative humid\ity and fed on the sugarcanes shoots (Rahlakar shoots (Rahlakar et alet al. 1972).. 1972).

• Larvae of the IVth Instar Stage were fixed at 50 Larvae of the IVth Instar Stage were fixed at 50 ºC for two minutes in the hot water (Jones 1962) ºC for two minutes in the hot water (Jones 1962) and pierced at the median dorsal line above the and pierced at the median dorsal line above the dorsal aorta to ooze out the required dorsal aorta to ooze out the required haemolymph and three slides were smeared with haemolymph and three slides were smeared with and fixed with methanol. and fixed with methanol.

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99 smears were prepared and 99 smears were prepared and counted counted after staining with after staining with Giemsa’s Giemsa’s

Stain. Stain.

The counts were first tabulated and The counts were first tabulated and relative percentage were calculated. relative percentage were calculated. Data were plotted in Histograms.Data were plotted in Histograms.

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Results and DiscussionsResults and Discussions

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Haemocytes were identified into Haemocytes were identified into six types following the method of six types following the method of (Jones, 1962) as follows:(Jones, 1962) as follows:

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Prohaeomocytes Prohaeomocytes

These are the smallest types of of These are the smallest types of of haemocytes. Their cytoplasm is strongly haemocytes. Their cytoplasm is strongly basophilic and forms a thin layer bound by a basophilic and forms a thin layer bound by a uniform clearly visible cell membrane uniform clearly visible cell membrane encasing comparatively large, spherical and encasing comparatively large, spherical and basophilic nucleus containing large compact basophilic nucleus containing large compact chromatin granules.chromatin granules.

The Cytoplasm of these cells varies from 7 – The Cytoplasm of these cells varies from 7 –

10um and nucleus is 4-7um with a 10um and nucleus is 4-7um with a cytoplasm nuclear area ratio (AC/AN) of 1.4 cytoplasm nuclear area ratio (AC/AN) of 1.4 ± 0.45 . ± 0.45 .

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PlasmatocytesPlasmatocytes

These form the bulk of the haemocyte These form the bulk of the haemocyte population population ≥≥70% and are comparatively 70% and are comparatively large, polymorphic cells with small, large, polymorphic cells with small, spherical to oval nuclei. These could be spherical to oval nuclei. These could be grouped into spherical, oval and irregular grouped into spherical, oval and irregular plasmatocytes.plasmatocytes.

The spherical , 5-8um in diameter with an The spherical , 5-8um in diameter with an

AC/AN ration of 3.1 ±102. The cytoplasm AC/AN ration of 3.1 ±102. The cytoplasm of these cells is generally vacuolatedof these cells is generally vacuolated

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Granular haemocytesGranular haemocytes

These are few, mostly spherical cells of These are few, mostly spherical cells of moderate size, oval shaped, 12-16um in moderate size, oval shaped, 12-16um in length and 11-14um in width with length and 11-14um in width with coparatively small, round eosinophilic coparatively small, round eosinophilic nuclei measuring 5-9um. nuclei measuring 5-9um.

Their cytoplasm is mildly basophilic with Their cytoplasm is mildly basophilic with few large, round eosinophilic granules few large, round eosinophilic granules and their AC/AN ration is 2.4±0.9 .and their AC/AN ration is 2.4±0.9 .

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Cystocytes Cystocytes

• These are also few and are rare similar These are also few and are rare similar to the granular haemocytes. They are to the granular haemocytes. They are 8-12um in diameter , but have one or 8-12um in diameter , but have one or more basophilic inclusions.more basophilic inclusions.

• Their nucleus is round 5-9um to oval Their nucleus is round 5-9um to oval

wit compactly arranged chromatin wit compactly arranged chromatin granules. The AC/AN ratio is 1.7 ± 0.6 .granules. The AC/AN ratio is 1.7 ± 0.6 .

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OenocytoidsOenocytoids

These are round or oval in shape with These are round or oval in shape with granular and strongly basophilic granular and strongly basophilic cytoplasm that has one or two vacuoles. cytoplasm that has one or two vacuoles. The nucleus more basophilic than The nucleus more basophilic than cytoplasm and eccentric in position. cytoplasm and eccentric in position.

These are 11-15um in diameter but there These are 11-15um in diameter but there are some oval ones which are 12-16um in are some oval ones which are 12-16um in length and 10-15 um in width. The AC/AN length and 10-15 um in width. The AC/AN ratio is 2.1±1.4 ratio is 2.1±1.4

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AdipohaemocytesAdipohaemocytes

• These constitute over 5% of the These constitute over 5% of the haemocytes in the haemolymph of weevil haemocytes in the haemolymph of weevil and moderate in size , 13-17um in length and moderate in size , 13-17um in length and 11-15um in width. and 11-15um in width.

• They contain very large fatty inclusions They contain very large fatty inclusions their AC/AN is 3.5 ± and 1.5. their AC/AN is 3.5 ± and 1.5.

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Mean value of the different types of haemocytes

Age/Day

Percentages of different haemocytes

Prohameocytes PlasmatocytesGranular haemocytes

Cystocytes Oenocytoids AdipohaemocytesMitosis

1 25.71±3.16 65.67±3.62 3.74±0.75 1.03±1.03 4.05±0.48 0.00 -----

2 14.39±3.59 56.30±6.54 4.88±2.22 1.74±1.18 9.08±2.15 13.65±4.30 -----

3 18.12±6.21 65.45±5.23 4.2±1.56 1.77±1.05 5.01±2.61 5.14±3.71 -----

4 11.2±5.82 74.24±3.79 5.12±3.89 1.05±1.52 3.19±2.60 3.76±3.76 -----

5 13.12±4.98 75.40±2.95 4.72±2.64 0.39±1.04 3.28±3.62 3.06±3.76 -----

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SummarySummary

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Duration between feeding and ecdysis is full of Duration between feeding and ecdysis is full of changes and developments including formation changes and developments including formation of the basement membrane. It is established of the basement membrane. It is established that the Plasmatocytes play a role in enhancing that the Plasmatocytes play a role in enhancing the growth and preparation of basement the growth and preparation of basement membrane. For the matter, the DHC in the IVth membrane. For the matter, the DHC in the IVth instar larvae may be of importance.instar larvae may be of importance.

In the larval stage the bulk of haemocytes is In the larval stage the bulk of haemocytes is

constituted with Plasmatocytes and constituted with Plasmatocytes and Prohaemocytes were the next in number and Prohaemocytes were the next in number and third main type remains Granular Haemocytes. third main type remains Granular Haemocytes. Remaining three types Cystocytes, Oenocyroids Remaining three types Cystocytes, Oenocyroids and Adipohaemocytes were rare in occurrence and Adipohaemocytes were rare in occurrence and densityand density..

Role of different types of haemocyte is discussed Role of different types of haemocyte is discussed in the preview of present findings on variations in the preview of present findings on variations amongst DHC of the larvae of different age. amongst DHC of the larvae of different age.

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