Giemsa Staining Haemocyte Types Detection

After I have reviewed the Haemocyte types in honey bee, therefor, I will provide you how apis mellifera haemocytes appear under the microscope, their basophilic, acidophilic colors and how Haemocytes Giemsa Staining  appear.

In this article, we will start from the beginning, from collecting haemolyph, passing through preparation of giemsa, consequently, ending up with detection of basicity and acidity of each type of haemocyte.

Hemolymph Collection from Apidae Adults

  • This is based on the research “A New Method for Quick and Easy Hemolymph Collection from Apidae Adults” by Grzegorz Borsuk *, Aneta A. Ptaszyńska , Krzysztof Olszewski , Marcin Domaciuk Patcharin Krutmuang Jerzy Paleolog.

  • Biological material used in the AMHS Methods

  • Immediately after emergence, Apis mellifera carnica workers and drones were individuallymarked with a paint marker and left inside their hive.
  • Upon reaching 10 days of age, 40marked workers and 40 marked drones were captured, placed in two separate cages, and transported to the laboratory for hemolymph sampling.
  • Hemolymph collection was performed immediately after the bees were delivered to the laboratory, no more than 30 minutes after their removal from their hive.
  • We also removed 40 individually caged virgin 4-day-old honeybee queens, each accompanied by five workers, from a colony lacking a nurse queen.
  • Hemolymph was collected from these queens within 30 minutes of their removal from the colony.
  • Of the 10 hemolymph samples taken from the queens, extracted by AMHS .
  • Steps for Haemolymph Collection from adult workers

  • Material required:

  • Gas burner.
  • Rectangular Styrofoam plate for holding an insect.
  • Tweezers.
  • An automatic pipette, pipette tips
  • 70% ethanol.
  • Cotton swabs.
  • Ice-bath to cool 0.2-mL Eppendorf tubes.
  • The equipment and laboratory room were sterilized with a UV lamp.
  • The procedure was performed in the immediate vicinity of a working gas burner to maintain sterile air.
  • MRS medium to confirm Hemolymph samples’ sterility.
  • Sabouraud medium to prove Hemolymph samples’ sterility.
  • Some information you should know:

  • WHY DO WE USE 70% ETHANOL NOT 100% or 90% or 95%?

  • Because you need also water to denature the proteins, enough water but not too much, not to dilute ethanol.
  • 95% ethanol evaporates very quickly at room temp, thus 70% is more effective.
  • 90% of alcohol has tendency to coagulate protein .Suppose the pure alcohol is poured over the bacterial cell .The alcohol will go through the cell wall of the bacteria in all directions ,coagulating the protein just inside the cell wall . So in this case the cell would get sealed & would become inactive but not dead.
  • 70% of alcohol, if it is poured over a bacterial cell, it also coagulates protein but at slow rate .So that it can get penetrated all over the cell. Now the entire cell is coagulated & the cell finally dies.
  • MRS Medium

  • Bacterial growth medium is so-named by its inventors: de Man, Rogosa and Sharpe.
  • MRS contains sodium acetate, which suppresses the growth of many competing bacteria.
  • Samples’ sterility.
  • Sabouraud Medium

  • Is named after,Raymond Sabouraud in 1892.
  • A type ofagar growth media containing peptones.
  • Used to cultivatedermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia.
  • The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth.
  • Samples’ sterility.
  • Method of the work

  • 40 Honey bee workers, 10 days age, large in size.
  • Hold the bee from its thorax on the Styrofoam plate.
  • take cotton swaps, merge it in 70% ethanol; disinfect the area around the antenna.
  • With the tweezers, detach the antenna, and then we’ll notice that a drop of haemolymph will appear.
  • Press on the abdomen to get large drop of the haemolyphm.
  • using the automatic pipette, collect the drops and put them in eppendorf tube.
  • The eppendorf tube will be placed immediately in an ice-bath (2±4EC) to prevent melanization.
  • Hemolymph sampled in this manner, could be immediately analyzed or frozen at −40EC for future studies (storage at −80EC is recommended if the storage period is to be longer than one month).
  • These samples placed on MRS medium to confirm their sterility.
  • Samples placed on Sabouraud medium to prove their sterility.

Why I preferred AMHS over other methods?

  • AMHS provide short time for collecting haemolymph that decrease the risk of melanizattion.
  • Low number of individuals used.
  • Larger insect size was associated with easier and faster hemolymph sampling, and with greater volumes of collected hemolymph.
  • In TCHS method, each of these procedures carries the risk that the ventriculus will be punctured, leading to sample contamination with intestine contents. Such contamination not only renders the sample unusable, but also requires that the capillary must be replaced with a new one or cleaned and disinfected before the next useذthus, increasing the overall hemolymph collection time.
  • Hemolymph sterility can also be compromised by puncturing the membrane connecting abdomen segments.
  • Moreover, hemolymph collection by decapitation may contaminate the hemolymph with nectar leaking from the bee’s stomach.
  • Ethanol disinfection and limited contact between the hemolymph drop and the bee body would ensure sample sterility.
  • The incubation of hemolymph acquired by AMHS on both MRS and Sabouraud agar mediums confirmed the hemolymph sterility.

Making a Peripheral Blood Smear

After we collected the blood sample, we will then prepare the blood smear so it will be ready for giemsa staining.

GIEMSA Staining

  • A Romanovsky type of stain named after German chemist and bacteriologist Gustav Giemsa, and is often used in histopathological diagnosis and cytogenetics.
  • Preparation

Supplement
  • Giemsa is a dye consisting of methylene blue-eosin and methylene blue. Named after Gustav Giemsa, a German chemist and bacteriologist.
  • This dye solution is used in histopathologic diagnosis.
  • It is used in staining blood films. Because of the basic and acidic dyes it contains it is favored in differentiating acid and basic granules in granulocyte.
  • Essential in determining the presence of negri bodies, Tunga species, spirochetes and other parasitic protozoans in blood.
  • Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow  Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta.
  • Used to visualize the classic “safety pin” shape in Yersinia pestis.
  • STAINING HAEMOLYMPH WITH GIEMSA STAINING

After we collect the haemolymph samples, we will stain the blood smear to know the acidity and basicity of each type of cells.

Watch the video

RESULTS

1- Plasmatocytes

Basophilic cytoplasm (blue color) and acidophilic nucleus (dark color)

Honey bee plasmatocytes-stained-with-Giemsa-cytoplasm-with-basophilic-characteristics-and-acidophilic-nucleus

2- Granulocytes

Acidophilic cytoplasm and basophilic nucleus 

Honey bee granulocytes stained with Giemsa and May-Grunwald; note cytoplasm with mainly acidophilic granules and basophilic nucleus

3- Oenocytoids

Whereas, Cytoplasm Acidophilic,we can find nucleus basophilic

4- Prohamocytes

Finally, Cytoplasm basophilic and nucleus acidophilic

Honey bee rohemocytes stained with Geimsa, basophilic cytoplasm and acidophilic nucleus

Stress-related Detoxifying Enzymes

In this part of my review, I have searched a lot about the detailed information about the effect of stress-related detoxifying enzymes, but with no gain.

So, I had two options, pain or gain but I have chosen the gain-pain, so, I decided to provide you, my blog readers, about these info, hoping you can understand this topic perfectly without suffering.

When we talk about stress-related detoxifying enzymes, then we are talking about the major groups including Glutathion-s-Transferase (GST), metallothionine like-proteins, phenoloxidase cascades, catalase, ctyochrome p450.

In every section, I will point to the origin, features, structure, mechanism and function of each item mentioned above.

Let’s start!!

 

 

 

 

 

Honey bee Haemocyte Types

HAEMOCYTE TYPES – FEATURES – FUNCTIONS – ORIGIN – HISTOLOGY

First of all, I thought if I’m looking for a cleared details about honey bee haemocyte types, one should find these information easily, but I was shocked when the true that one cannot get them from one place or even an article yet.

Hence, I have wasted my precious time meaningless, couldn’t make any progress in this part, especially, that wasn’t what I thought.

In addition, while my research is about Honey bee Haemocyte types, I have found very the same researches that made me confused and disappointed as I wanted the basics and the concepts that can help me in my Master.

As a result, I have decided to include all what I have learnt and gained from information and other stuff in this post.

In this article, I will not sum up but will write in details with images exposed by numerous researchers that helped me in my research, but I have ordered them and an information from here to there while trying to understand what materials I have.

Finally, therefore, I have put them in detailed points as they will be easy to read and understand.

Honey bee Plasmatocytes Characteristics

Honey bee Irregular shape of plasmatocyte with bilobulate nucleus with phagocytosis process

  • Shape
  •  Irregular plasmatic membrane.
  • Diversity in shape, may be ovoid, spherical.
  • Rare Pseudopodia.

Honey bee Irregular-shaped-plasmatocytes-with-multilobulate-nucleus

  • Cytoplasm

  • Large amount.
  • No cytoplasmic inclusions
  • Low density
  • Numerous polyribosomes
  • Little developed RER.
  • Well-developed mitochondria.
  • Few vesicles.
  • Basophilic, blue color in geimsa staining.ribosomes (R), and vesicles (VE) in the cytoplasm.

  • Nucleus

  • Small
  • Centric, exocentric, oval, spherical
  • Chromatin
  • Acidophilic, dark color in geimsa staining.
  • 0.2 size to cytoplasm

Honey bee plasmatocytes-stained-with-Giemsa-cytoplasm-with-basophilic-characteristics-and-acidophilic-nucleus

  • Function

  • Initiate phagocytosis that lead to encapsulation and nodule formation that synthesize desmosomes-like junctions.
  • Difference between lamellocytes and plasmatocytes

  • Circulatory haemolymph: lamellocytes are few where plasmatocytes are numerous
  • Monolayers: lamellocytes are flattened in both monolayer and fixed blood, where plasmatocytes are ovoid and become flattened in capsule-nodule formation.

Honey bee Granulocytes Characteristics

Honey bee Granulocyte Oval cell with lobulate exocentric nucleus (N) and the presence of mitochondria

  • Shape

  • Regular plasmatic membrane.
  • Diversity in shape, may be ovoid, spherical.
  • Thin-large pseudopodia.
  • Phagolysosomes-like inclusions.Honey bee Granulocytes with lobulate nucleus
  • Cytoplasm

  • Large amount.
  • Low density granulations.
  • No cytoplasmic inclusions
  • Numerous polyribosomes
  • Well-developed RER with flocculant material
  • Well-developed mitochondria.
  • Few vesicles.
  • Numerous pinococytes vesicles.
  • Acidophilic, pink color in geimsa staining, but occasionally basophilic (bluish) granulationsHoney bee granulocytes stained with Giemsa and May-Grunwald; note cytoplasm with mainly acidophilic granules and basophilic nucleus
  • Nucleus

  • Small
  • Centric, exocentric, oval, spherical
  • Chromatin found rupture formed in packets
  • Basoophilic, dark color in geimsa staining.
  • 0.3 size to cytoplasm
  • Function

  • Phagocytosis.
  • In Drosophila, they are named Drosophila plasmatocytes.
  • They don’t form bulk capsules around invaders so that they get rid of them, but they are the first cells to attach to the pathogen cells at the first of capsule-nodule formation.
  • According to some authors, the EXCOCYTOSIS of granulocytes serves to attract plasmatocytes to build capsule or nodules around the invader.
  • In monolayers, they don’t even spread extensively, but remain spherical, adhering to coverslip with lamellipodia and filopodia.
  • ORIGIN : LYMPH GLANDS

  • NOTE THAT: Drosophila plasmatocytes like Lepidopterian Granulocytes and both lake typical inclusions.

Honey bee Oenocytoids Characteristics

  • Shape

  • Crystal cells
  • Large cells
  • Irregular plasmatic membrane.
  • Diversity in shape, may be ovoid, spherical.
  • Crytalline-like inclusions, paracrystalline.

Honey bee Oenocytes has more electrodense cytoplasm and irregular nucleus

  • Cytoplasm

  • Low amount.
  • Homogenous
  • High density
  • Irregular cytoplasmic inclusions
  • sharp contour-like crystalline inclusions.
  • Short and enlarged RER .
  • Few vesicles.
  • Acidophilic in Geimsa Stain
  • Nucleus

  • Large
  • Centric, exocentric andspherical
  • Chromatin found in packets
  • Function

  • Melanization (melanin synthesize)
  • One of phenoloxidase cascades found in these cells.
  • In monolayers, fragile and easy to lyse shortly after haemolymph collection in absence of fixation.
  • ORIGIN : Haematopeotic organs.

Honey bee Prohamocytes Characteristics

  • Shape

  • Irregular plasmatic membrane.

 

  • Cytoplasm

  • Small amount.
  • Homogenous
  • Little developed RER.
  • Basophilic, geimsa staining.

y bee Honeprohemocyte irregular shape and the spherical nucleus (N) occupying a large part of the cytoplasm.

  • Nucleus

  • Very large
  • Centric, exocentric, oval, spherical
  • Acidophilic, in geimsa staining.

Honey bee rohemocytes stained with Geimsa, basophilic cytoplasm and acidophilic nucleus

  • Function

  • Thought to be the precursor to differential haemocyte types.

Sources@

Blood cell

HEMOCYTES OF THE COCHINEAL INSECT: ULTRASTRUCTURE

HAEMOCYTE TYPES AND TOTAL AND DIFFERENTIAL COUNTS IN UNPARASITIZED AND PARASITIZED HELICOVERPA ARMIGERA LARVAE

Honey bee humoral immune response “apis mellifera”

Honey bee humoral immune response

  • The recognition of invading pathogen either as bacteria or fungi or even viruses is followed by the immediate de novo synthesis of antimicrobial peptides (AMPs) and their secretion into the hemolymph (Zasloff, 2002; Bulet et al., 2004).
  • These peptides are mainly synthesized by the fat body and in a lesser degree by the hemocytes, integument, gut, salivary glands and reproductive structures (Nappi and Ottaviani, 2000).
  •  The humoral immune response is based on the products of characterized immune genes induced by microbial infection and encode antimicrobial peptides, which are synthesized predominantly in fat body and released into hemolymph (Hoffmann,1995; Gillespie et al., 1997; Nakatogawa et al.,2009; Shia et al., 2009).
    • Hemocytes and epithelial layers of the integument and the gut are also sites for the synthesis of such molecules. These genes are either not expressed or are constitutively expressed at a low rate prior to infection (Hoffmann,1995; Engstrِm, 1998).
    • In addition, humoral immune responses include activation of enzymic cascades that regulate coagulation and melanization of hemolymph, and production of reactive oxygen and nitrogen species (ROS-RNS)(Gilespie et al., 1997; Bogdan et al., 2000; Nappiand Vass, 2001; Hoffmann, 2003; Mavrouli et al.,2005).

What are the steps of humoral immune response in honey bee?

steps of Honey bee humoral immune response
steps of humoral immune response in honey bee @ http://www.biologyexams4u.com/

  • Step 1: When a naïve B cell interacts with an antigen specific for its surface antibody, it gets activated and starts dividing rapidly. The process is called clonal selection. The phenomenon of selective proliferation of B cells in response to their interaction with the antigen is called clonal selection.
  • Step 2: Remember, B cell activation and proliferation depends on the cytokinins secreted by TH cells. In this interaction, B cells itself function as antigen presenting cells or APCs which present antigen on its surface Class II MHC molecules. This Class II MHC molecule with antigen on T cell surface interacts with T-cell receptor or TCR of TH cells. In addition CD40 of B cells interacts with CD40L of TH cells. This interaction activates B cells to differentiate and proliferate.

 

 

  • Activated B cells differentiates into 2 types of cells
  • 1) An antibody producing effector cells or plasma cells. Plasma cells do not have a surface Ig. Plasma cells secrete antibodies. A single plasma cell can secrete ~ 2000 antibody molecules/second.
  • 2) Memory B cells which has long life span and possess a surface Ig and responsible for immunologic memory.
  • Thus humoral immunity involves the production of antibodies by plasma cells and this constitutes the primary immune response.
  • When there is an exposure to the same antigen second time, the memory B cells are alerted and rapidly differentiate into plasma cells that produces antibodies. This constitutes the secondary immune response.

 

·         Antimicrobial peptides

    • Over 150 antimicrobial peptides (AMPs) have been isolated and characterized in insects. These molecules are small, 12-50 amino acids, cationic peptides, which bind anionic bacterial or fungal membranes leading to disruption and cell death(Zasloff, 2002; Yount and Yeaman, 2006).
    • Although they have different structure and target organisms(bacteria or fungi), the AMPs are classified in four groups; a) cecropins, b) cysteine-rich peptides, c)proline-rich peptides, and d) glycine-rich peptides.
    • Cecropins were firstly isolated in cecropiaafter injection with bacteria (Hultmark et al., 1980;Steiner et al., 1981). These peptides are producedin response to septic injury by either Gram positive or Gram negative bacteria bacteria and affect cellular proliferation by inhibiting the synthesis of proteins of the cell membrane.
    • Defensins and drosomycin are cysteine-richpeptides. Defensins, destroy mostly gram-positive peptides by forming channels in the plasma membrane which leads to cell lysis, while drosomycin has an antifungal activity.
    • Diptericin is an antibacterial peptide that has been found only indiptera species and is induced upon Gram negative bacteria infection in a way similar to attacines (Nappiand Ottaviani, 2000). Lysozymes are enzymes that degrade peptidoglycans of the bacterial cell wall.They are also found in other animals, plants, fungi and bacteriophages (Bulet et al., 1999).

 

       Enzymic cascades

       Coagulation of hemolymph

  • Apis mellifera like other insects that have developed mechanisms for the coagulation of hemolymph, in case of wounding, to prevent loss of body fluids (Theopold et al., 2002).In the cockroach Leucophaea maderae, hemocytes secrete a calcium dependent transglutaminase that catalyzes the polymerization of lipophorins and vitellogenin-like proteins. These last proteins have a domain homologous to the Von Willebrand clotting factor in mammals (Βohn et al., 1994).
  • The most characterized mechanism is the one in Lymulus polyphemus, which appears to be similar in Drosophila (Vimlos and Kurucz, 1998). According to this, LPS and â-1,3-glucan trigger a serine protease chain reaction, finally leading to the coagulation of the hemolymph. In addition, serine protease activates melanization cascade (Nappi et , al., 1995;Mavrouli et al., 2005; Sideri et al., 2007).
  • It must be noted the dual role of serine protease in the insect immunity since intermediate metabolites of these two cascades, preclotting enzymes, melanin derivatives and reactive oxygen species, are toxic invading pathogens.

        Melanization of hemolymph

  • Melanization, the pathway leading to melan information, has a central role in defense against a wide range of pathogens and participates in wound healing as well as in nodule and capsule formation in some lepidopteran and dipteran insects, (Lavineand Strand, 2001; Lavine and Strand, 2003;Mavrouli et al., 2005).
  • Melanization depends on tyrosine metabolism. Briefly, tyrosine is converted to dopa, an important branch point substrate, by activated phenoloxidase (PO).
  • Dopa may be either decarboxylated by dopa decarboxylase (Ddc) to dopamine or oxidised by PO to dopa quinone. Dopamine is also an important branch point substrate, because dopamine-derived metabolites either via PO or through other enzymes are used in several metabolic pathways, participating in neuro transmission, cuticular sclerotization, cross linking of cuticular components via quinone intermediates, phagocytosis, wound healing and melanization in immune reactive insects (Fearon,1997;Aderemand Underhill,1999; LingandYu,2005;Marmarasand Lampropoulou,2009).

        Characterisation of dopamine receptors in insect (Apis mellifera) brain

  • Dopamine has been detected in the nervous system of many insect species, and enzymes required for dopamine synthesishave also been identified. Dopamine-sensitive adenylate cyclase activity in the insect brain indicates that at least some of the actions of dopamine are mediated through the elevation of intracellular cAMP, but the identity of the receptors involved has yet to be resolved.
  • Dopamine receptors in the vertebrate brain have for some years, been classified into two major subtypes, D1and D 2 receptors respectively. With the advent of molecular cloning techniques, however, additional subtypes have been revealed. To date, five pharmacologically distinct dopamine receptor subtypes have been recognized. Two of these cloned receptors (D 1and Ds), exhibit functional and pharmacological properties that resemble classical D 1 dopamine receptors, whereas the other three (D 2, D 3 and D 4) are D2-1ike in their pharmacological characteristics.

 

Honey bees haemocytes nanoparticles “Apis mellifera”

Honey bees haemocytes nanoparticles “Apis mellifera”

Honey bees haemocytes nanoparticles
Honey bees haemocytes nanoparticles
  1. Introduction

In this model, we will use Drosophila melanogaster as it is homologous.

1.1. What is difference between homologous and heterologous gene?

Heterologous gene expression is inserting or synthesis of a gene into a host cells that it normally does not produced while homologous gene expression is the over expression of a gene in the same host that it normally produces .

Source @ J.E. Inetianbor @ researchgate.net

 

Homologous or very similar to genes implicated in mammalian innate immune defences’’ (Hoffmann, 2003).

Source @ Carlos Ribeiro, Michel Brehe @ Insect haemocytes: What type of cell is that?

 

1.2. What is heterologous gene expression?

Heterologous expression refers to the expression of a gene or part of a gene in a host organism, which does not naturally have this gene or gene fragment. Insertion of the gene in the heterologous host is performed by recombinant DNA technology.

Source @ Heterologous expression – Wikipedia

1.3. What’s the difference between autologous, homologous (allogenic), and heterologous blood transfusions?

Autologous blood transfusion is the collection and re-infusion of the patient’s own blood or blood components. Homologous, or more correctly allogenic, blood transfusions involves someone collecting and infusing the blood of a compatible donor into him/herself. Heterologous blood transfusions are those that involve someone infusing blood and its components from a different species. Withdrawal, storage and preservation, and re-infusion of blood products are carefully regulated in the health industry because infusion of infectious, old, or incorrectly matched blood could result in hospitalization and possibly death.

Source @ What’s the difference between autologous, homologous (allogenic …