Immune response in the larva of the dung beetle Phyllognathus excavatus against human blood cells as foreign bodies

2.1 Identification

The identification of the dung beetle was conducted based on characteristics and diagnostic keys available in the literature (original descriptions): (Forster, 1771; Janssens, 1942; Paulian, 1941), and in comparison with the Phyllognathus species depicted in the following papers (Al-Ali et al., 2015; Errouissi et al., 2009; Márquez-Rodríguez, 2016).

2.2 Sampling

Numerous larvae of the scarab beetle Phyllognatus excavatus (Coleoptera: Scarabaeidae) were collected from rural zone (west southern of Tunisia) and reared in moistened soil in 10 L plastic tanks at ambient temperature. Larvae reaching the last instar (L3) were selected for the assays. The hemolymph was obtained by pricking the larvae with needles.

2.3 Light and scanning electron microscopy analysis in larval hemocytes

For light microscopy, pooled of hemolymph was collected by syringe after previously perfusing the thorax of larva with heparin as anticoagulant (Grégoire, 1953). Hemolymph dropped on glass slides and allowed to settle for some minutes in natural air condition. Cells were fixed in methanol for 10 min. Hemocytes were then stained with May-Grunewald-Giemsa stain and slides were quickly washed with demineralized water. After room temperature, the slides were dehydrated with ethanol.

To support the light microscopy and to know the exact sizes and shapes of certain cells, scanning electron microscopy was used. For SEM, hemolymph was obtained by picking the larvae with needles and placed directly on fragments of slides (Richardson et al., 2018). These were then allowed to dry in room temperature for some minutes and carried out under SEM.

To investigate the reaction of hemocytes on human blood, a drop of blood was added to the hemolymph placed on slides and the same steps described above were followed.

2.4 Hematology analysis

So that, to more improve results obtained under microscopy, automated hematology analyzes were carried out. Samples were processed in the Mindray BC-3600 analyzer. Thus, to investigate whether larval hemolymph contains small cells (lymphocytes-like) and platelets-like, hemolymph sample was collected in EDTA tube and analyzed using the automated analyzer. To estimate the rate and to ensure the type of eliminated cells after mixing larval hemolymph and human blood, a mixture of 2 mL of hemolymph with 2 mL of blood was softly agitated in an EDTA tube for some seconds and then brought to the analysis using the automated. A complete blood count of a healthy adult man was performed using the automated as a reference to compare the results. Proposed in vitro screening of insect immune pathway and mechanistic testing is shown in Fig. 1.

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Fig. 1

Proposed experimental set-up indicates the immune pathway induced by the scarabid beetle larvae hemocytes. HRBC: human red blood cells; Gran: Granulocytes; PLT-Like: Platelet-Like; Plasm: plasmatocytes; Cogul: coagulocytes..

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3.1 Circulating cells in P. excavatus larval hemolymph

In this study, different circulating cells in the hemolymph of the P. excavatus larva were observed under both light microscopy and scanning electronic microscopy (SEM).

Insect hemolymph presents numerous hémocytes types that are mainly identified using their functional, morphological, and histochemical characteristics. The characterization of the hemocytes drew the attention of the researchers for a long time and made the object of various classifications. The classification of insect hemocytes varies among species and differs from author to another. While hemocyte types described in literature do not occur in all insects, the five most recognized and common types are plasmatocytes, granulocytes, oenocytoids, prohemocytes, and spherulocytes (spherule cells) (EL Sadawy et al., 2020; Lavine and Strand, 2002; Ling and Yu, 2005; Strand, 2008). In mosquitoes, Hillyer and Christensen (2002) classified circulating hemocytes of adult Aedes aegypti (Diptera, Culicidae) into granulocytes, oenocytoids, adipohemocytes and thrombocytoids. Other studies based on previous investigations classified hemocytes from A. aegypti into only granulocytes, oenocytoids and prohemocytes (Strand, 2008). Culex pipiens quinquefasciatus transfers many important diseases, such as West Nile virus, lymphatic filariasis and Japanese encephalitis (Da Silva et al., 2000). Despite its epidemiological importance, authors contradict each other in the classification of hemocytes. Brayner et al. (2005), Brayner et al. (2007) divided circulating hemocytes of this vector into six different types. In contrast, in a more recent review, hemocytes in this mosquito are conclusively thought as are four types: prohemocytes, oenocytoids, plasmatocytes and granulocytes (Wang et al., 2011). In hymenoptera, two type of circulating hemocytes were identified in the hemolymph of the worker honeybee larva (Apis mellifera L.) by phase contrast and transmission electron microscopies. These were the plasmatocytes and granulocytes. The ultrastructural investigation of the bee hemocytes revealed the possible presence of a third type of cells, which has been called “macrophage” by analogy with vertebrate macrophages (Belaid et al., 2013). Galleria mellonella larva (Lepidoptera, Pyralidae) has been widely used as a model to investigate the virulence of several human pathogens. Recent study undertaken by Wu et al. (2016) allowed the recognition of four types of hemocytes (plasmatocytes, granular cells, spherule cells and oenocytoides) in the hemolyph of the wax moth. In comparative studies on the immunity defense mechanisms in larvae and adults of Cetonischema aeruginosa (Coleoptera, Scarabaeidae), the ultrastructure of the circulating cells in the third instar larval stage has been explored using both light and transmission electron microscopy. The different hémocytes found in the larval hemolymph were identified as granulocytes prohemocytes, plasmatocytes, coagulocytes, oenocytoids and spherulocytes (Giulianini et al., 2003).

In our study, four different hemocytes where observed by the microscopy in the hemolymph of the third instar larval Phyllognathus excavatus. They were identified as plasmatocytes, granulocytes, oenocytoides, and coagulocytes. Granulocytes appear oval in shape with short pseudopodia (∼15 µm in diameter) (Fig. 2A, E). Oenocytoids present a round shape containing dense granules (Fig. 2B). Plasmatocytes were the most observed type of hemocytes. They appear to be elongate, plate, and irregular in shape (∼15 µm in lentgh) (Fig. 2C, D). Coagulocytes (Fig. 2E, F) are oval in shape and very small (3–5 µm) comparing with the other hemocyte types.

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Fig. 2

Circulating cells in the hemolymph of P. excavatus. Granulocytes observed by LM (A) and SEM (E) (scale bar = 5 µm); Oenocytoides observed by LM (B); Plasmatocytes observed by LM (C) (scale bar = 5 µm) and SEM (D) (scale bar = 20 µm); Coagulocytes detected by SEM (E, F) (scale bar = 5 µm).

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3.2 Immune defense in P. excavatus larva

In our study, it seems that larval hemolymph contain a large number of small cells (coagulocytes). As seen in Fig. 2F, the SEM suggested the involvement of these cell type circulating in the scarab larval into the hemolymph coagulation (promote clot formation by extruding threadlike, cytoplasmic extension, and granules). It seems that coagulocytes circulating in the hemolymph of the larva of P. excavatus as autonomous drones for hemostatic and immune surveillance share remarkable functional similarities with mammalian blood platelets. In mammals, platelets are the smallest cell type in circulating blood, averaging only from 2.0 to 5.0 μm in diameter, 0.5 μm in thickness, and having a mean cell volumes between 6 and 10 femtoliters. They are subcellular fragments released from megakaryocytes that circulate in blood as anucleate and discoid cells. The average human body has in the order one trillion circulating blood platelets (200 000/µL), each platelet has an average lifespan of 8–10 days. One hundred billion new platelets must be produced daily from bone marrow megakaryocytes to maintain platelet counts between 150 × 10⁹ and 400 × 10⁹ platelets per liter of whole blood (Thon and Italiano, 2012). Their small size and discoid shape result in their being pushed to the vessel edge by blood flow (Hartwig, 2007), allowing them to flatten over the damaged surface rapidly. Despite their small size and anucleate structure, platelets play diverse roles in many physiological processes. Platelets also have an important impact on immune cell extravasation due to their adhesive receptors and their high abundance in the blood (Boilard, 2017). They are also known as the cellular mediator of thrombosis (Morrell et al., 2014). Among their main functions, platelets respond to blood vessel damage by secreting the contents of their granules and aggregating to form a platelet clot (pand-aids) (Thon and Italiano, 2012). They are healers that release growth factors and other soothing molecules that help damaged tissue to rebuild (Leslie, 2010). As evident from the studies of many researchers, platelets are also soldiers that trigger the protective response known as inflammation, alert immune cells, and even attack microbial invaders (Avecilla et al., 2004; Larson and Watson, 2006; Thon and Italiano, 2012).

In order to determine the different hemolymph cells involved in the immune defense against foreign bodies, a mixture of larval and human blood in vitro shows the hemolysis of red blood cells by coagulocytes. As detected in Fig. 3A, a trigger of coagulocytes having almost the same dimension with blood cells (5 µm) in the hemolysis of the surrounding foreign human red blood cells. It is suggested that this cell type are also able to kill invaders (not only involved in the clot formation). Moreover, plasmatocytes (in cluster and individual) and granulocytes (Fig. 3B–E) were revealed to be professional foreign bodies’ killer in Phyllognathus excavatus larvae. Thus, upon their exposure to human blood cells, they are able to recognize foreign surfaces and secrete soluble mediators in the vicinity that serve to pathogens phagocytosis and lysis. Additionally, results in our study showed that platelets-like figured in Fig. 2F, 3C and counted in Table 1, may well play a great role in distinguishing foreign crops and immune system defense. On this basis (the larval hemocytes attack human red blood cells as foreign bodies), it is suggested that beetle larval hemolymph can also be used to combat unwanted bodies in humans, such as cancer cells, and in other therapeutic applications. But this needs further studies.

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Fig. 3

Hemolysis of red blood cells by larval small cells (A). Granulocyte, platelets-like and coagulocytes (B, C) and plasmatocytes (D, E) detected in the Phyllognathus excavatus hemolymph involved in the cellular immune reaction. PLT-Like: Platelet-Like; RBC: red blood cells.

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Table 1 Lymphocytes-like and platelets-like count of scarabid beetle Phyllognathus excavatus larva. PLT: Platelets; WB: white blood; RB: red blood; Lymph: Lyphocytes; Gran: Granulocytes; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; RDW-CV: red blood cell distribution width-coefficient variation; RDW-SD: red blood cell distribution width-standard deviation; MPV: mean platelet volume; PDI: platelet distribution index; PCT: Plateletcrit; fl: femtoliters; µl: microliters; dL: deciliter; ml: milliliters, pg: pictograms.

Element analysis		Result	Unit	Element analysis		Result	Unit
WB	R	10.0	103/µL	MCH		*****	pg
Lymph#	RH	8.0	103/µL	MCHC		*****	g/dL
Mid#		*****	103/µL	RDW-CV		*****
Gran#		*****	103/µL	RDW-SD		*****	fL
Lymph#	RH	0.801		PLT	L	26	103/µL
Mid#		*****		MPV		9.0	fL
Gran#		*****		PDI	H	18.5
RB	L	0.0	106/µL	PCT	L	0.23	mL/L
HGB	L	0.0	g/dL	P-LCC	L	8	109/L
HCT	L	0.0	%	P-LCR		0.302
MCV		*****	fL

On the viewpoint of immune events, more investigation under the automated hematology analyzer confirms that the hemolymph of the study larva contain a large number of lymphocytes-like and platelets-like. It seems that the larval coagulocytes were considered as lymphocytes by the automated analyzer, as they share almost the same characters. As showed in Table 1 the content of the larval hemolymph in coagulocytes (lymphocytes-like) reach 8 × 10⁶/µL. The hemogram showed also the presence of platelets-like (∼26 × 10³/µL) circulating in the larval hemolymph. It is assumed that among the different hemolymph cells, vast number of platelets-like derived from coagulocytes (Fig. 2f) was present, which readily fragmentize playing practically the same role (clot formation and immune defense). As well as, their contribution in invaders attack was shown (Fig. 3C). Notably, the notion of platelets-like in invertebrates is not new. When studying the hemolymph cells of the mollusk, Incilaria fruhstorferi (Gasteropoda: Pulmonata), Furuta et al., 1990; Furuta and Yamaguchi, 2011) found that the largest slug native of Japan possesses three types of hemolymph cells: macrophage-like, lymphocyte-like and fibroblast-like.

Further, a mixture of larval hemolymph with human blood showed the destruction of approximately 3/4 of the human red blood cells as showed in Table 2 under the larval immune system compared with a complete red blood count of a healthy adult man (Table 3). Fig. 4 depicts the results. Fig. 4A shows that the larval hemolymph contains lymphocytes-like but with higher content than that of human blood. The mixture increased the content of this type of cell. Fig. 4C shows that the larval hemolymph presents platelets-like but with lower content than that of human blood. Fig. 4B revealed two main notes: (i) the larval hemolymph is devoid of red blood cells. (ii) the content of human red blood cells was reduced by 75 % when mixed with larval hemolymph (∼4.06 × 10⁶/µL vs. ∼ 0.58 × 10⁶/µL) with a significant difference (p < 0.01). This can be explained in fact only red blood cells were considered as foreign crops by the larval immune system (coagulocytes, plasmatocytes, granulocytes, and platelets-like) and then the majority was killed. Thus, insect hemocytes recognize foreign bodies and phagocytose pathogens in a manner similar to that of mammalian leukocytes (Browne et al., 2013), just as insects and maamals share innate immune responses (Krautz et al., 2014).

Table 2 Effect of the larval hemolymph in the decrease of human red blood cells.

Element analysis		Result	Unit	Element analysis		Result	Unit
WB	RH	12.7	103/µL	MCH	RH	181.0	pg
Lymph#	RH	7.3	103/µL	MCHC	RH	142.9	g/dL
Mid#	RH	1.8	103/µL	RDW-CV	RH	0.194
Gran#	R	3.6	103/µL	RDW-SD	RH	120.5	fL
Lymph#	RH	0.576		PLT	L	78	103/µL
Mid#	R	0.141		MPV	H	12.6	fL
Gran#	RL	0.283		PDI	H	18.8
RB	RL	0.58	106/µL	PCT	L	0.98	mL/L
HGB	L	10.5	g/dL	P-LCC		39	109/L
HCT	RL	7.3	%	P-LCR	H	0.500
MCV	RH	126.7	fL

Table 3 Normal red blood count of a healthy adult man.

Element analysis	Result	Unit	Element analysis		Result	Unit
WB	6.2	103/µl	MCH		30.0	pg
Lymph#	2.0	103/µl	MCHC		31.6	g/dL
Mid#	0.6	103/µl	RDW-CV	L	0.170
Gran#	3.6	103/µL	RDW-SD	H	51.2	fL
Lymph#	0.328		PLT	L	134	103/µL
Mid#	0.090		MPV	H	8.5	fL
Gran#	0.582		PDI	H	15.9
RB	4.06	106/µL	PCT	L	1.14	mL/L
HGB	12.2	g/dL	P-LCC		35	109/L
HCT	38.7	%	P-LCR	H	0.260
MCV	95.2	fL

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Fig. 4

Histograms showing the variation of some elements figured in the larval hemolymph and human blood compared to a mixture of human blood with larval hemolymph. Lymphocytes (A); Red blood cells (B); Platelets (C).

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