# Comparative assessment of hemolymph composition and bioactivity in different bee species

Salma A Elfar, Iman M Bahgat, Mohamed A Shebl, Mathieu Lihoreau,
Mohamed M Tawfik

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Salma A Elfar, Iman M Bahgat, Mohamed A Shebl, Mathieu Lihoreau, Mohamed M Tawfik. Comparative assessment of hemolymph composition and bioactivity in different bee species. The Journal of Basic and Applied Zoology, 2025, 86 (1), pp.100. 10.1186/s41936-025-00520-y. http://hal-05381951

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# Comparative assessment of hemolymph composition and bioactivity in different bee species

Salma A. Elfar¹ ID, Iman M. Bahgat¹ ID, Mohamed A. Shebl² ID, Mathieu Lihoreau³ and Mohamed M. Tawfik¹* ID

**Abstract**

**Background** Hemolymph, as mediator of immune responses and nutrient circulation, can be used as physiological marker of an insect's health, environmental quality or ecological adaptations. Recent studies reported intraspecific variation in protein contents and biological activities of the hemolymph of honey bees related to their diet. Here we measured interspecific variation in three common bee species in the Mediterranean Basin with contrasting ecologies: *Apis mellifera*, *Megachile sicula*, and *Xylocopa pubescens*.

**Results** Despite all the bees were collected in the same area, we found important quantitative and qualitative variations of hemolymph extracts across species. Samples of *A. mellifera* and *M. sicula* had much higher protein concentration, anticancer, antimicrobial and antioxidant activities than samples of *X. pubescens*.

**Conclusions** This descriptive study suggests life history traits of bee species have strong influences on their hemolymph properties and call for future large scale comparative analyses across more species and geographical areas.

**Keywords** Honey bees, Carpenter bees, Megachilids, Proteome, Antiproliferative, Antibacterial

**Introduction**

Bees are among the most ecologically and economically important insect groups, including over 20,000 species categorized in seven families (Michener, 2007). Beyond their vital role in pollination, many bee species can produce biologically active substances including honey, pollen, wax, royal jelly and venom. These products exhibit

antioxidant, antibacterial or antitumor activities (Kolayli & Keskin, 2020). Among the most studied species: *Apis mellifera* honey bees which are studied for honey, wax, royal jelly, propolis and venom production (Alvarez-Suarez, 2025), *Apis cerana* that is studied for its medicinal honey and adaptability to local environments (Yancan et al., 2019), stingless bees that produce unique honeys with high bioactive compound content, especially in tropical regions (Al-Hatamleh et al., 2020) and *Bombus spp.* (bumblebees) that are investigated for their pollen and antimicrobial peptides (Simmons & Angelini, 2017). While bee-derived products like honey and venom have long attracted scientific interest for their therapeutic properties, recent attention has shifted toward hemolymph, a vital circulatory fluid with promising biomedical potentials. Recent studies showed interest to

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Springer

<footnote>*Correspondence:
Mohamed M. Tawfik
tawfik@sci.psu.edu.eg
¹Zoology Department, Faculty of Science, Port Said University, Port Said, Egypt
²Department of Plant Protection, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt
³Research Center on Animal Cognition, Center for Integrative Biology, CNRS, University Paul Sabatier, 31062 Toulouse, France</footnote>
bee hemolymph, which is a vital fluid equivalent to the blood in vertebrates and plays essential role in circulation of nutrients and immune agents to the infected tissue (Ashrafuzzaman et al., 2022).

Recently, the FDA approved the treatment of viral skin infection with products from blister beetles. Insects used in medicine offer a valuable reservoir of bioactive molecules that support various health applications (Anal et al., 2025).

Insect hemolymph is a multifunctional circulatory fluid that plays a vital role in the immune defense actions. It contains hemocytes—specialized immune cells responsible for main immunological processes such as phagocytosis, encapsulation, lysis of invading organisms and also the secretion of humoral proteins. Additionally, peptides derived from insect hemolymph exhibit a broad spectrum of therapeutic activities, including antimicrobial, anticancer, and antiviral effects, highlighting its potential as a source of bioactive compounds for biomedical applications. (Gamal et al., 2023). These properties allow the insect hemolymph to be a promising source for pharmaceutical and agricultural applications.

Bee hemolymph contains various hydrophilic (e.g. hemocyanin) and hydrophobic (e.g. apolipophorins) proteins that contribute to immune actions and physiological regulation (Dhawan et al., 2017; Rothemund et al., 1999; Schäfer et al., 2021). High concentrations of these proteins improve the immune responses of bees and their resistance to infections (Schulz et al., 2019; Skowronek et al., 2021; Strachecka et al., 2015).

Increasing evidence suggests that hemolymph also possesses interesting biological properties including anticancer and antioxidant properties (Elfar et al., 2023; Mokarramat-Yazdi et al., 2021; Sojka et al., 2016; Strachecka et al., 2022).

So far, most research on bee hemolymph has been performed in honey bees (Ararso et al., 2018; Barragán et al., 2015; Bogaerts et al., 2009; Chan et al., 2006; Horvatinec & Svečnjak, 2020). Although recent work described significant intraspecific variations in the proteomic profiles of the hemolymph of bees that are likely attributed to the diet proving that the artificially fed bees relying on sucrose solution without pollen showed the lowest protein concentrations and the weakest biological activities, including cytotoxic, antimicrobial, and antioxidant properties when compared to bees that fed on diverse natural resources (Barragán et al., 2015; Elfar et al., 2023), little is known about the interspecific variations within the bee taxa. This gap in the comparative data limits our understanding of the variations of hemolymph composition among bee species and addressing this gap is important for expanding our knowledge of the application of bee hemolymph in biomedical and ecological research.

The bee hemolymph demonstrated promising potential in biomedical research due to its rich composition of immune cells, enzymes, and antimicrobial peptides. These components contribute to innate defense actions and have shown therapeutic properties such as antibacterial, antiviral, and anticancer activities. For instance, the activity of phenoloxidase and hemocyte profiles in honey bees is linked to enhanced immune responses when supplemented with probiotic diets, this suggests possible applications in immunomodulation and drug development (Robino et al., 2024). In ecological research, bee hemolymph serves as a valuable physiological biomarker for monitoring environmental stress and colony health. Variations in hemolymph composition across different bee species can reflect exposure to pollutants, nutritional status, and disease susceptibility. This makes the hemolymph analysis a beneficial tool for monitoring ecosystem dynamics and a sessing how human-induced environmental changes affect pollinator species and their ecological stability (Elfar et al., 2023).

Therefore, it has been proposed that the proteomic structure of hemolymph can be used to monitor the physiological conditions of bees as well as the quality of their environment. Since bees are found across a wide range of habitats worldwide (Michener, 2007), we also expect hemolymph variation across species to exist and reflect key ecological adaptations.

Here we explored interspecific variations in hemolymph properties of bees from the Mediterranean Basin. We analysed hemolymph samples of three common species collected in the same area in Egypt: *Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*. *A. mellifera* (honey bees) are social Apidae nesting in cavities (Conroy & Holman, 2022). *X. pubescens* (carpenter bees) are solitary Apidae that build their nests in wood (Vicidomini, Vicidomini, 1997; von Reumont et al., 2022). *M. sicula* are solitary Megachilidae that build their nests with mud (Kronenberg & Hefetz, 1984). This is a descriptive study that primarily aimed at analyzing honey bee hemolymph and comparing it to that of other common yet poorly studied bee species. and studying the species-specific traits for allowing better understanding of the hemolymph diversity in bees. This study enhances our understanding of immune system diversity by analyzing hemolymph composition across three ecologically distinct bee species, revealing species-specific traits with promising implications for both environmental assessment and biomedical research. The hemolymph samples were collected using two complementary solvents and analyses were performed to quantify their proteomic content as well as their potential anticancer, antibacterial, antioxidant, and hemolytic activities.
# Materials and methods

## Bee sample collection

We sampled bees from three bee species with a sweep net (Shebl & Farag, 2015) between October 2020 (A. mellifera) and February 2021 (X. pubescens and M. sicula) in cultivated locations planted with faba beans (Vicia faba) in the Ismailia Governorate area (Egypt). We collected all specimens from each species in the same location on the same day. We then stored the bees at -20 °C for 5 min to assist extraction and analysis of the hemolymph (Tutun et al., 2020) (Details of the collected bee specimens and the extracted hemolymph Table 1).

## Hemolymph extraction and preparation

We extracted the hemolymph from the specimen by using sterile insulin syringes to puncture the body close to the membrane of the coxa and applying a slight pressure on the abdominal region. We pooled the hemolymph samples from the same bee species and kept them in sterile Eppendorf tubes at -20 °C until lyophilization by using freeze drying (Tutun et al., 2020). We then dissolved the weighed lyophilized hemolymph samples in two different but complementary solvents (a hydrophilic solvent: phosphate buffer saline—PBS (Nugraha et al., 2021); and a hydrophobic solvent: dimethyl sulfoxide—DMSO (Arakawa, 2018)) in order to extract a maximum of molecules as the polarity of the solvent influences the protein structure, solubility and stability (Pace et al., 2004). For each test, both of PBS and DMSO were used as negative controls.

## Analysis of the hemolymph protein content

### Protein concentration

We measured the protein concentration (mg/mL) of the hemolymph extracts following Desjardins et al. (2009) using a Thermo Scientific™ (Waltham, MA, USA) Nano Drop™ One Micro volume UV-Vis Spectrophotometer with Bovine serum albumin (BSA) as standard in each sample (2 µL, 1 mg/mL). We analyzed three samples per bee species.

<table><caption>**Table 1** Details about bee sampling and hemolymph extraction</caption><thead><tr><td>Bee species</td><td>Body length (mm)</td><td>Number of specimens</td><td>Weight of lyophilized hemolymph (grams)</td><td>Amount of hemolymph (microliter)</td></tr></thead><tbody><tr><td>Apis mellifera</td><td>10–15</td><td>81</td><td>0.15</td><td>~800</td></tr><tr><td>Xylocopa pubescens</td><td>13–30</td><td>20</td><td>0.34</td><td>~1700</td></tr><tr><td>Megachile sicula</td><td>15–20</td><td>29</td><td>0.106</td><td>~590</td></tr></tbody></table>

## SDS-polyacrylamide gel electrophoresis

We used gel electrophoresis to separate the protein bands of the hemolymph extracts based on their molecular weight using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) following Laemmli (1970) with modifications. We mixed equal volumes of hemolymph samples with solubilizing buffer (62.5 mM Tris-HCl (pH 6.8), 20% glycerol, 25(w/v) SDS, 0.5% 2-mercaptoethanol and 0.01% bromophenol blue). After heating for 4 min at 95 °C, we inserted the samples into the wells (15 µL sample with 100 µg/mL protein concentration per well) of the separating gel (12% PAGE gel). We then ran an electrophoresis at constant 35 mA for 2 h using Consort N.V. (Belgium) mini vertical electrophoresis system with running buffer. We prepared the staining gel using 0.1% Coomassie Brilliant Blue (R-250) for the visualization of the protein bands.

## High performance liquid chromatography (HPLC)

In complement to the electrophoresis, we performed an HPLC and analyzed the chromatograms by investigating the protein peaks and their separation based on their retention time. We obtained equal concentration (5 mg/mL) of hemolymph extracts in each of the two solvents (PBS or DMSO). We analysed the extracts (70 µL) using a YL9100 HPLC System with Stationary phase C18 column (Promosil C18 Column 5 µm, 150 mm × 4.6 mm) with acetonitrile (ACN) gradients of (10–100%) acetonitrile in water mobile phase for 50 min at flow rate = 1 mL/min. The chromatogram was detected using a UV detector at wavelength 280 nm following Basseri et al. (2016) with some modifications.

## Analysis of biological activities

### Antiproliferative activity by MTT assay

We measured antitumor activities of hemolymph extracts using a 3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay (Mosmann, 1983). Cells from human liver cancer (HepG2) and human cervical cancer (HeLa) were obtained from the Holding company for biological products and vaccines (VACSERA), Giza, Egypt. After that, the cells were proliferated in RPMI-1640 medium provided with 10% fetal bovine serum (FBS) and 1% penicillin (antibiotic), and then we incubated the cells for growth in 5% CO₂ at 37 °C.

We seeded the cells in 96-well plate for 24 h (5 × 10³ cells/well). After incubation, we treated the cells with 100 µL of the hemolymph extracts at serial concentrations in PBS and DMSO solvents (31.25, 62.5, 125, 250, 500 and 1000 µg/mL) and incubated them at 37 °C in 5% CO₂ atmosphere for 48 h. We then washed the cells using PBS. We added fresh medium with MTT dye and incubated at 37 °C for 4 h. We then added DMSO for the
solubilization of the formazan crystals in the viable cells (Constante et al., 2022). We used a Bio-Tek ELISA micro plate reader to measure the absorbance at 540 nm. The experiment was performed in triplicates. We calculated the percentage of the cell viability (%) as follows:

$$ \text{Cell viability (\%)} = (A_T / A_C) \times 100 $$

where A_T was the absorbance of treated cells with extracts, and A_C was the absorbance of the control cells (untreated cells). The IC₅₀ values (i.e. concentration of extracts that cause inhibition in the growth of 50% of the cells) were calculated for each sample using a dose-response curve with dose concentration (X-axis) and cell cytotoxicity percentage (Y-axis).

### Antibacterial activity using agar well diffusion

We analyzed the antimicrobial activity of the hemolymph using the agar well diffusion method of Magaldi et al. (2004) against four bacterial strains (*Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli* and *Salmonella typhimurium*). We inoculated the agar plate surface by distributing bacterial suspension. We then made a 6.0 mm hole aseptically using a sterilized tip and added 100 µL of each extract (10 mg/mL) or control into the well. We used PBS and DMSO as negative controls and Gentamycin (20 µg/mL) as a positive control. After the incubation, we expressed the in vitro antimicrobial activity as inhibition zones in millimeters (mm) (Espinel-Ingroff et al., 2007, 2011).

### Scavenging ability using DPPH free radicals

We measured the scavenging ability of hemolymph extracts using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical (Braca et al., 2001). We incubated a mixture of 100µL DPPH methanolic solution (0.004% in 95% methanol) and 300 µL of each hemolymph extracts at concentration 1 mg/mL and standard at 25 °C in the dark for about 30–60 min. We used ascorbic acid (Vitamin C) as positive control, and measured the color changes using a spectrophotometer at 515 nm. We calculated the DPPH scavenging activity of the samples using the following equation (Hasan et al., 2009):

$$ \text{Antioxidant activity (\%)} = 100 \times [(A_N - A_E) / A_N] $$

where A_N was the absorbance of the negative control, and A_E was the absorbance of the sample or of the standard.

### Hemolytic activity assay

We assessed the hemolytic activities of the bee hemolymph extracts against human erythrocytes by applying procedures of Malagoli (2007). We added blood samples

to test tubes containing blood anticoagulant (EDTA) and centrifuged the tubes for 5 min at 10,000 rpm. We then suspended red blood cells in sterile PBS and incubated them with 100 µL volume from series of various concentrations of the tested extracts (range: 156.25–5000 µg/mL). After incubation of the tubes at room temperature for one hour, we centrifuged the tubes for 5 min at 1 × 10³ rpm and measured the absorbance of the supernatant at 570 nm. We used Triton 10% as positive control while PBS and DMSO 10% as negative controls. We ran triplicate analysis for each bee species. We then calculated the hemolysis percentage for each extract as follows:

$$ \text{Hemolysis (\%)} = 100 \times [(A_S - A_N) / (A_P - A_N)] $$

where A_S was samples absorbance, A_N was the negative control absorbance, and A_P was the positive control absorbance.

### Statistical analysis

We analyzed the data in SPSS 22.0. We used Student's t-tests to compare protein concentrations and IC₅₀ of the hemolymph extracts in PBS and DMSO. We compared parameters of bee hemolymph (i.e. protein concentrations, IC₅₀s, antibacterial and antioxidant activities in either PBS or DMSO) across species using one-way ANOVAs followed by Tukey's HSD post-hoc tests. We used two-way ANOVAs to compare parameters of bee hemolymph (i.e. protein concentrations and antioxidant activities) in both solvents among the three bee species. While we used two-way ANOVAs to compare parameters of bee hemolymph (i.e. IC₅₀s and antibacterial) in both solvents among the three bee species against each one of the tested cell lines or bacterial strains independently. We considered significant differences between samples when the p-value was lower than 0.05. All means are reported with their standard error (mean ± SE).

## Results

### Analysis of protein content

#### Protein concentration

Protein concentration of hemolymph varied across species and solvents (Two-ways ANOVA, significant effect of species (F (2,12)=111.97, P<0.001), a significant effect of solvent (F (1,12)=20.41, P<0.001) and a significant species x solvent: F (2, 12)=45.62, P<0.001; Fig. 1). The highest protein concentration was recorded for *M. sicula* in PBS (0.27±0.01 mg/mL, n=3). Lowest protein concentrations were recorded for *X. pubescens* in PBS (0.02±0.005 mg/mL, n=3) and DMSO (0.06±0.001 mg/mL, n=3). Hemolymph of *A. mellifera* had values falling in between for PBS and similar high values as *M. sicula* for DMSO.
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Protein concentration in mg/mL for hemolymph extracts of three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) in PBS or DMSO. Bars and error bars show the mean ± SE of triplicate results. Values with different lower-case letters in a column indicate significant differences between extracts (Tukey's HSD test: $P \le 0.05$). *SDS-polyacrylamide gel electrophoresis*</figcaption></figure>

### SDS-polyacrylamide gel electrophoresis

Protein bands in electrophoresis gels of hemolymph extracted in PBS and DMSO had a molecular weight ranging from ~5 to 250 kDa (Fig. 2).

The PBS dissolved extracts were characterized by five bands common to the three bee species with molecular weights of ~40, ~60, ~70, ~110 and ~250 kDa (Fig. 2A). Other bands however were only observed in some species. A band with molecular weight of ~7 kDa was only recorded in *A. mellifera* and *M. sicula*. Another band with molecular weight of ~10 kDa was only recorded in *A. mellifera* and *X. pubescens*. Five bands with molecular weights of ~4, ~5, ~17, ~37 and ~100 kDa were

exclusively recorded in *M. sicula*. The DMSO dissolved extracts were characterized by five bands common to the three bee species with molecular weights of ~5, ~17, ~40, ~60 and ~110 kDa (Fig. 2B). A band with molecular weight of ~150 was recorded in *A. mellifera* and *X. pubescens*. Two bands with molecular weights of ~10 and ~25 kDa were only recorded in *X. pubescens*.

### RP-HPLC analysis

By analyzing the chromatogram of the PBS dissolved hemolymph extracts we found the presence of 6 peaks common to the three bee species at retention times of 7.5, 11.1, 14.4, 15.4, 22.9 and 28.3 min (Table 2). In DMSO dissolved extracts, we detected 10 peaks common to the three bee species at retention times of 9.5, 23.3, 24.6, 25.5, 26.8, 28.3, 32.6, 33.2, 40.3 and 44.8 min (Table 2). RP-HPLC chromatograms are available in the supplementary Figures S1 and S2.

### Analysis of biological activities

#### Antiproliferative activity

We evaluated the antiproliferative actions of the hemolymph extracts against the viability of the in vitro propagated hepatic and cervical carcinoma cells. All hemolymph extracts resulted in inhibition of the cell viability against the tested cancer cell lines in a dose-dependent manner after 48 h of incubation, irrespective

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Pictures of SDS-PAGE gel for hemolymph of the three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) extracted in A PBS or B DMSO. M is the marker</figcaption></figure>
<table><caption>**Table 2** HPLC most common chromatogram peaks profiles of three bee species hemolymph</caption><thead><tr><th rowspan="3">Retention time (minutes)</th><th colspan="4">Peak area (× 10<sup>2</sup> mV.s)</th></tr><tr><th colspan="2">A. mellifera</th><th colspan="2">X. pubescens</th><th colspan="2">M. sicula</th></tr><tr><th>PBS</th><th>DMSO</th><th>PBS</th><th>DMSO</th><th>PBS</th><th>DMSO</th></tr></thead><tbody><tr><td>7.5</td><td><strong>4.6</strong></td><td>N.A</td><td><strong>0.7</strong></td><td>N.A</td><td><strong>177.4</strong></td><td>N.A</td></tr><tr><td>9.5</td><td>N.A</td><td><strong>327</strong></td><td>N.A</td><td><strong>7.7</strong></td><td>N.A</td><td><strong>9.7</strong></td></tr><tr><td>11.1</td><td><strong>2.3</strong></td><td>N.A</td><td><strong>0.4</strong></td><td>N.A</td><td><strong>25.2</strong></td><td>N.A</td></tr><tr><td>14.4</td><td><strong>5.9</strong></td><td>N.A</td><td><strong>0.3</strong></td><td>N.A</td><td><strong>10.3</strong></td><td>N.A</td></tr><tr><td>15.4</td><td><strong>5.5</strong></td><td>N.A</td><td><strong>0.4</strong></td><td>N.A</td><td><strong>2.2</strong></td><td>N.A</td></tr><tr><td>22.9</td><td><strong>13.9</strong></td><td>N.A</td><td><strong>1.2</strong></td><td>N.A</td><td><strong>3.5</strong></td><td>N.A</td></tr><tr><td>23.3</td><td>N.A</td><td><strong>3.6</strong></td><td>N.A</td><td><strong>0.1</strong></td><td>3.6</td><td><strong>3.4</strong></td></tr><tr><td>24.6</td><td>N.A</td><td><strong>3.4</strong></td><td>N.A</td><td><strong>0.6</strong></td><td>N.A</td><td><strong>1.4</strong></td></tr><tr><td>25.5</td><td>9.1</td><td><strong>3.6</strong></td><td>1.5</td><td><strong>0.1</strong></td><td>N.A</td><td><strong>3.8</strong></td></tr><tr><td>26.8</td><td>N.A</td><td><strong>4.0</strong></td><td>1.1</td><td><strong>0.3</strong></td><td>2.8</td><td><strong>0.6</strong></td></tr><tr><td>28.3</td><td><strong>5.3</strong></td><td><strong>1.5</strong></td><td><strong>1.5</strong></td><td><strong>0.4</strong></td><td><strong>1.2</strong></td><td><strong>0.7</strong></td></tr><tr><td>32.6</td><td>N.A</td><td><strong>1.4</strong></td><td>N.A</td><td><strong>2.2</strong></td><td>N.A</td><td><strong>1.0</strong></td></tr><tr><td>33.2</td><td>N.A</td><td><strong>0.3</strong></td><td>N.A</td><td><strong>1.3</strong></td><td>N.A</td><td><strong>0.8</strong></td></tr><tr><td>40.3</td><td>N.A</td><td><strong>0.1</strong></td><td>N.A</td><td><strong>0.1</strong></td><td>N.A</td><td><strong>0.1</strong></td></tr><tr><td>44.8</td><td>N.A</td><td><strong>1.0</strong></td><td>N.A</td><td><strong>1.2</strong></td><td>N.A</td><td><strong>0.9</strong></td></tr></tbody></table>

The hemolymph of three bee species (Apis mellifera, Xylocopa pubescens and Megachile sicula) extracted in PBS or DMSO

<figure><img src="image_6.png" /><figcaption>**Fig. 3** Effects of hemolymph extracts of three bee species (Apis mellifera, Xylocopa pubescens and Megachile sicula) on cell proliferation of human cancer (HepG2 and HeLa) cell lines at different concentrations in PBS or DMSO</figcaption></figure>

of the solvent used (Fig. 3). However, the DMSO dissolved extracts showed higher overall cytotoxic activity than the PBS dissolved extracts for HepG2 and for HeLa when using two-way ANOVAs to compare IC₅₀s of bee

hemolymph in both solvents among the three bee species against each one of the tested cell lines independently.

(Two-ways ANOVA (solvent x species), HepG2: F (2, 12) = 26.667, P < 0.001; HeLa: F (2, 12) = 117.111, P < 0.001) (Fig. 4).
<figure><img src="image_7.png" /><figcaption>**Fig. 4** IC₅₀ in µgram/mL of hemolymph extracts of three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) against HepG2 and HeLa cell lines, using PBS or DMSO as solvents. Bars and error bars represent the mean values (±SE) of triplicate results. Different lower-case letters indicate significant differences between extracts (Tukey's HSD test: P ≤ 0.05)</figcaption></figure>

<table><caption>**Table 3** Inhibition zone of three bee species hemolymphs against various types of bacteria</caption><thead><tr><th rowspan="2">Sample</th><th colspan="3">PBS</th><th colspan="3">DMSO</th><th rowspan="2">CON.</th></tr><tr><th>A. mellifera</th><th>X. pubescens</th><th>M. sicula</th><th>A. mellifera</th><th>X. pubescens</th><th>M. sicula</th></tr></thead><tbody><tr><td>B. subtilis</td><td>34 ± 1.0</td><td>26 ± 0.3</td><td>26 ± 0.8</td><td>32 ± 1.6</td><td>25 ± 0.8</td><td>27 ± 1.4</td><td>22 ± 0.6</td></tr><tr><td>S. aureus</td><td>25 ± 2.0</td><td>25 ± 0.8</td><td>23 ± 1.4</td><td>24 ± 0.9</td><td>25 ± 0.3</td><td>22 ± 0.5</td><td>15 ± 0.9</td></tr><tr><td>E. coli</td><td>40 ± 1.5</td><td>24 ± 1.4</td><td>25 ± 0.5</td><td>38 ± 1.0</td><td>22 ± 0.8</td><td>23 ± 0.3</td><td>17 ± 1.0</td></tr><tr><td>Salmonella typhimurium</td><td>N.A</td><td>25 ± 1.1</td><td>24 ± 1.1</td><td>N.A</td><td>21 ± 0.8</td><td>22 ± 1.1</td><td>23 ± 0.7</td></tr></tbody></table>

The inhibition zones in (mm) of hemolymph from three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) extracted in PBS or DMSO against various types of bacteria (*Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli* and *Salmonella typhimurium*). CON: Gentamycin as positive control, N.A: not applicable, ± S.E.: standard error values obtained from triplicate measurements.

Among the DMSO dissolved extracts, *M. sicula* hemolymph had the highest cytotoxic effects against HepG2 and HeLa cell lines (IC₅₀ HepG2 = 77.35 µg/mL, IC₅₀ HeLa = 101.2 µg/mL). The lowest cytotoxic effects were recorded for *A. mellifera* hemolymph (IC₅₀ HepG2 = 153.1 µg/mL, IC₅₀ HeLa = 148.46 µg/mL) (Fig. 4). Among the PBS dissolved extracts, *A. mellifera* hemolymph had the highest cytotoxic effects (IC₅₀ HepG2 = 649.4 µg/mL, IC₅₀ HeLa = 312.54 µg/mL) and *X. pubescens* had the lowest (IC₅₀ HepG2 = 669.2 µg/mL, IC₅₀ HeLa = 447.2 µg/mL) (Fig. 4).

### Antimicrobial activity

We observed the antibacterial activity of hemolymph extracts against Gram-positive and Gram-negative bacteria (Table 3, see supplementary material in FigureS3). The results showed comparable antibacterial activities of hemolymph extracts among the tested bee species and solvent; *Bacillus subtilis* (ANOVA, F (2, 12) = 0.800, P = 0.472), *Staphylococcus aureus* (ANOVA, F (2, 12) = 0.042, P = 0.960), *Escherichia coli* (ANOVA, F (2, 12) = 2.054, P = 0.171) and *Salmonella typhimurium* (ANOVA, F (2, 12) = 2.771, P = 0.102).

<figure><img src="image_8.png" /><figcaption>**Fig. 5** DPPH radical scavenging activity of hemolymph extracts of three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) extracted in either solvent PBS or DMSO. Bars and error bars denote the mean values (± S.E.) of triplicate results. Different lower-case letters indicate significant differences between extracts (Tukey's HSD test: P ≤ 0.05)</figcaption></figure>

### DPPH radical scavenging assay

The hemolymph extracts of *A. mellifera*, *X. pubescens* and *M. sicula* possessed effective scavenging actions in both PBS and DMSO (Fig. 5). Overall, the hemolymphs of the different species showed different antioxidant activities (two-ways ANOVA, species: F (2, 12) = 5.06, P = 0.025) and these values were higher in PBS dissolved
<figure><img src="image_9.png" /><figcaption>**Fig. 6** Hemolytic activity of hemolymph extracts from three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) at different concentrations against human erythrocytes. Hemolymph samples were extracted in PBS or DMSO</figcaption></figure>

<table><caption>**Table 4** Summary of analysis of hemolymph of three bee species</caption><thead><tr><th rowspan="2"></th><th colspan="2">A. mellifera</th><th colspan="2">X. pubescens</th><th colspan="2">M. sicula</th></tr><tr><th>PBS</th><th>DMSO</th><th>PBS</th><th>DMSO</th><th>PBS</th><th>DMSO</th></tr></thead><tbody><tr><td>Protein concentration</td><td>(±)</td><td>(±)</td><td>(–)</td><td>(–)</td><td>(+)</td><td>(±)</td></tr><tr><td>Anticancer activity</td><td>(+)</td><td>(–)</td><td>(–)</td><td>(±)</td><td>(±)</td><td>(+)</td></tr><tr><td>Antimicrobial activity</td><td>(+)</td><td>(+)</td><td>(±)</td><td>(±)</td><td>(±)</td><td>(±)</td></tr><tr><td>Antioxidant activity</td><td>(+)</td><td>(±)</td><td>(–)</td><td>(–)</td><td>(±)</td><td>(+)</td></tr><tr><td>Hemolytic activity</td><td>(–)</td><td>(–)</td><td>(–)</td><td>(–)</td><td>(–)</td><td>(–)</td></tr></tbody></table>

Three bee species (*Apis mellifera*, *Xylocopa pubescens* and *Megachile sicula*) extracted in two solvents (PBS and DMSO). (+): high, (±): medium and (–): low.

extracts (two-ways ANOVA, solvents: F (1, 12) = 10.729, P = 0.007). However, for a given species the antioxidant activity in both solvents was comparable (two-ways ANOVA, species x solvent: F (2, 12) = 2.451, P = 0.128).

The highest antioxidant activity was reported for *A. mellifera* PBS extracts (29.1%) and *M. sicula* DMSO extracts (25.2%), while *X. pubescens* reported the lowest antioxidant activity (25.7%) in PBS and (19.3%) in DMSO (Fig. 5).

### Hemolytic activity

Hemolytic properties of the bee hemolymphs were tested against human erythrocytes. For the three bee species, hemolymph extracts possessed either no or low hemolytic activity in reference to negative (PBS (0% hemolysis) and DMSO 10% (4% hemolysis)) and positive (Triton10% (99% hemolysis)) controls (Fig. 6).

### Discussion

We aimed to evaluate the interspecific variability in hemolymph protein content and biological activities from bees collected in the same locality in Northern Egypt by comparing domestic *A. mellifera* to the two much less studied wild species *X. pubescens* and *M.*

*sicula*. We found considerable variation among the three bee species (see summary results in Table 4).

Overall, *X. pubescens* hemolymph possessed the lowest protein concentrations, *A. mellifera* hemolymph had intermediate values, and *M. sicula* hemolymph showed highest protein concentrations irrespective of the solvent used for extraction. Previous studies reported intra-specific variations in hemolymph protein concentration primarily correlated to the diet of bees, even among bees from the same worker caste (Basualdo et al., 2013; El Mohandes et al., 2010; Elfar et al., 2023; Zheng et al., 2014). Since all our samples came from the same study site and were collected on the same plants, this suggests *A. mellifera*, *X. pubescens* and *M. sicula* had significantly different diets. Bee diet varies based on various parameters (Wright et al., 2018) including the physiological states of the bees themselves and the developmental stages of larvae they need to feed (Barraud et al., 2022; Sedivy et al., 2011; Williams, 2003). Another source of variation is the impact of presence of phoretic mites on *Xylocopa* species that feeds on pollen paste (Watmough, 1973), the main source of proteins in the hemolymph of bees (Frias et al., 2016). Another study showed that even if the three bee species visited the same plant source
there is still variations in the durations of the visits which for sure affects their feeding habits (Mohamed et al., 2024).

The lowest protein concentrations in *X. pubescens* were associated with the weakest antioxidant and antibacterial activities against both *B. subtilis* and *E. coli*. This was also associated with the weakest anti-proliferative activities among the PBS dissolved hemolymph extracts against both HepG2 and HeLa cell lines. By contrast, the highest protein concentrations of *M. sicula* in both solvents were associated with the strongest anti-proliferative activities among DMSO dissolved hemolymph extracts against both tested cancer cell lines. This is consistent with the fact that high protein concentration in the bee hemolymph is associated to high resistance to pathogens, increased life span, and improved individual immunity (Schulz et al., 2019; Skowronek et al., 2021; Strachecka et al., 2015).

SDS-PAGE gel analysis revealed proteomic profiling of the hemolymph extracts of the three bee species in PBS or DMSO solvents. Protein bands common to the three species, with molecular weight (4–5 kDa) in DMSO dissolved hemolymph were similar to cercopin, a family of small proteins have been isolated from different insect hemolymph which possesses anti-bacterial properties against both Gram positive and Gram negative bacteria (Keppi et al., 1986; Steiner et al., 1981). Extractions in DMSO also revealed another protein bands common to the three species that were detected at molecular weight (~17 kDa) similar to a group of lectins (Viswambari Devi et al., 2010). Lectins are defense proteins that can participate in many biological activities such as antimicrobial, antioxidant and anticancer in arthropods. It can be also considered as natural anticancer agent and contribute in immune actions (Pathak, 1993; Fujii et al., 2012; Cheung et al., 2015; Xia et al., 2018; Pinto et al., 2019; Zakzok et al., 2021; Eleftherianos et al., 2021). CLIPC9 similar bands were observed at (~37 kDa) in both PBS and DMSO extracts consistent with their detection in the hemolymph of *Anopheles gambiae* (Sousa et al., 2020). CLIP proteases are serine proteases found in the hemolymph of all insects and participate in the innate immune responses. The protein bands with molecular weight (~40 kDa) were also found in both PBS and DMSO extracts are in the same range of lipopolysaccharide recognition protein (LRP). LRP was purified from the plasma of large beetle larvae, *Holotrichia diomphalia* with immune roles as it participates in agglutinating activities against *E. coli* and other bacteria (Ju et al., 2006). Protein bands detected at molecular weight (~60 kDa) in PBS or DMSO extracts are within the range of purified protein fractions extracted from cockroach hemolymph with potent antimicrobial activities (Basseri et al., 2016). Moreover, common detected protein bands at (~70 kDa) were similar

to hemocyanin, an oxygen transporter protein that possesses antioxidant, antiparasitic, antimicrobial, anticancer and other biotic activities. This protein is found in most arthropods (Guo et al., 2011; Zakzok et al., 2021, 2022).

The HPLC chromatograms of all the hemolymph samples extracted in PBS exhibited some common peaks with the DMSO dissolved extracts. Remarkably, some peaks were only common between two species only, the highest peak areas were recorded among the PBS dissolved extracts from *M. sicula* reflecting greater protein content. This result is consistent with the total protein concentrations, which shows that the proteomic content of *M. sicula* PBS samples.

The variety and likely dominance of the bioactive proteins such as cercopins, lectins, CLIP proteases, LRP and hemocyanin in PBS and DMSO hemolymph extracts may clarify the effective biological activities of hemolymph in the current study including the antimicrobial, anticancer and antioxidant activities. Generally, DMSO dissolved hemolymph extracts possessed higher cytotoxic activities against HepG2 and HeLa cancer cell lines than the effect of PBS extracts. Our proteomic profiling analysis revealed the presence of variations according to the type of dissolved proteins in different solvents, in agreement with studies on the impact of solvent on the protein structure (Amer et al., 2019; Nugraha et al., 2021). According to Kramer et al. (2012) and Nugraha et al. (2021), altered solvents affects the protein solubility thus may influence the hemolymph bio-activities. Hydrophobic proteins such as lipoprotein complexes were used as delivery vehicles for anticancer drugs (Lacko et al., 2002).

The fact that none of the hemolymph extracts possessed lytic activities against erythrocytes agrees with the results of a previous study on the hemolytic activities of honey bee hemolymph (Elfar et al., 2023). These results may encourage the possible participation of bee hemolymph extracts in safe and selective therapeutic agents. Accordingly, bee hemolymph mixed with herbal extracts exhibited high anticancer efficiency with less or no hemolytic activities (Mokarramat-Yazdi et al., 2021).

Honey bees have innate immune mechanisms including the physical barriers and both humoral and cellular actions for their defense against infections and pathogens that affects the bee immune system and thus affects the bee health and the social behavior of these insects develops their social immunity which reduces the stress of the individual immune response of the bees (Larsen et al., 2019). Further studies are required on the immune responses of other different bee species for explaining the variations in their proteomic content and biological activities.
## Conclusions

Our study highlights significant interspecific variability in the protein concentration and biological activities of the hemolymph of three common Mediterranean bee species, likely related to variation in their life history traits. Broader scale comparative studies, using similar procedures as ours, across different geographical areas are now needed to investigate variations among bee species and how they are related to their ecology and evolution.

### Abbreviations

<table><tr><td><i>A. mellifera</i></td><td><i>Apis mellifera</i></td></tr><tr><td><i>M. sicula</i></td><td><i>Megachile sicula</i></td></tr><tr><td><i>X. pubescens</i></td><td><i>Xylocopa pubescens</i></td></tr><tr><td>PBS</td><td>Phosphate buffer saline</td></tr><tr><td>DMSO</td><td>Dimethyl sulfoxide</td></tr><tr><td>BSA</td><td>Bovine serum albumin</td></tr><tr><td>SDS-PAGE</td><td>Sodium dodecyl sulphate polyacrylamide gel electrophoresis</td></tr><tr><td>ACN</td><td>Acetonitrile</td></tr><tr><td>MTT</td><td>3-[4,5-Methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide</td></tr><tr><td>HepG2</td><td>Human liver cancer</td></tr><tr><td>HeLa</td><td>Human cervical cancer</td></tr><tr><td>FBS</td><td>Fetal bovine serum</td></tr><tr><td>DPPH</td><td>1,1-Diphenyl-2-picrylhydrazyl</td></tr><tr><td><i>B. subtilis</i></td><td><i>Bacillus subtilis</i></td></tr><tr><td><i>S. aureus</i></td><td><i>Staphylococcus aureus</i></td></tr><tr><td><i>E. coli</i></td><td><i>Escherichia coli</i></td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s41936-025-00520-y.

### Supplementary material 1.

Supplementary material 2. Figure S1. RP-HPLC chromatogram of PBS dissolved hemolymph extracts of different bee species. Figure S2. RP-HPLC chromatogram of the DMSO dissolved hemolymph extracts of different bee species. Figure S3. Antimicrobial activity of hemolymph from three bee species (Apis mellifera, Xylocopa pubescens and Megachile sicula) extracted in PBS or DMSO against E. coli bacteria and by using negative control. Table S1. A list of collected bee species for hemolymph extraction for the study.

### Acknowledgements

We would like to thank Prof. Soliman Kamel and Haj. Saied Aboud for their contribution in the bee sample collection.

### Author contributions

MT, IB and MS: Conceptualization and Supervision. SE, MT, IB and MS: Methodology, Software, validation, formal analysis, investigation, resources and data curation. SE, MT, IB, MS and ML: Writing—Original Draft Preparation, Writing—Review & Editing, visualization. All authors read and approved the final manuscript.

### Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

### Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

## Declarations

### Ethics approval and consent to participate

The ethical approval was obtained from the Research Ethics Committee of the Faculty of Agriculture, Suez Canal University, Egypt (Approval No. 73/2022). All procedures were performed in accordance with institutional ethical standards and guidelines.

### Consent for publications

Not applicable.

### Competing interests

The authors declare no competing interests.

Received: 19 July 2025 / Accepted: 15 November 2025

Published online: 25 November 2025

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