Introduction

Insect resources have been used by human beings for a long time as medicine. Medicinal insects and their products can be used to treat many different diseases either directly or indirectly. In traditional medicine, insect bodies, eggs, and secretions have been used to cure diseases for more than 2000 years (Farghaly and Sadek 2020).

Insects make up more than 55% of the total biodiversity, representing an important natural source of antimicrobial peptides (AMPs) and anticancer peptides (ACPs) (Faruck et al. 2016). An essential feature of their success in life is possessing the ability to evolve in many situations and thrive in environments replete with potentially parasitic and pathogenic competitors (Ahamad et al. 2012). This happened by evolving defensive mechanisms against any stress (Diamond et al. 2009). So, insects offer a source of natural compounds that may have valuable therapeutic utility against human diseases (Hasaballah et al. 2019).

Cellular defense in insects is provided by hemolymph cells or hemocytes. The hemocytes serve as the major cellular immune response, triggering cytophagocytosis and melanization tubercle formation (Wu et al. 2015). So, when the insects were infected by the invading cells, the pattern recognition proteins/receptor (PRPs) in insects would firstly recognize, and then initiate the activation and regulation of a series of innate immune response (Ohta et al. 2016). Among insects, dipterans are known to respond to any threat by producing various humoral defense proteins (Boman et al. 1992; Wojda 2017). The flies grow and survive in dirty places, so it is believed that they have strong antimicrobial peptides (Ahamad et al. 2012).

Insects and their products are essential ingredients in the preparation of drugs in folk medicine (Fred-Jaiyesimi and Awobajo 2011). The housefly, Musca domestica Linnaeus, 1758 is the most common of all domestic flies, extracts from houseflies have shown as antioxidant (Shittu et al. 2014), antimalarial (Shittu et al. 2013), antibacterial, antiviral, antitumor and immune activation properties (An et al. 2004). Antibacterial peptides have been isolated from the larval hemolymph of flesh fly; Sarcophaga peregrina Robineau-Desvoidy, 1830 larvae (Okada and Shunji 1983). Biomphalaria alexandrina Ehrenberg, 1831 snails serve as valuable animal models across a spectrum of scientific disciplines due to their unique biological features, ease of maintenance, and relevance to various research areas. The versatility of snails as experimental subjects has facilitated advancements in fields such as neurobiology, ecology, pharmacology, developmental biology, physiology, genetics, and evolutionary studies (Ibrahim et al. 2023b). Biomphalaria alexandrina snails having high reproductive capacity, and ability to adapt to different environments (Adekiya et al. 2020).

The ecological adaptability of snails has made them essential in ecological and environmental research. Studies on snail populations provide insights into biodiversity, habitat preferences, and the impact of environmental changes, making them important bioindicators in freshwater ecosystems. Many studies used B. alexandrina snails as an accepted invertebrate models to elucidate the toxic effect of different material (Abdel-Tawab et al. 2022; Ibrahim et al. 2023b; Morad et al. 2022; Moustafa et al. 2018). Biomphalaria, known for its accessibility, ease of collection, adaptability to laboratory conditions, sensitivity to water, and chemical pollutants, stands out as an ideal candidate for laboratory monitoring in ecotoxicological studies and the analysis of multiple biomarkers (Ruppert et al. 2017). This species has been widely utilized as a paradigm in various scientific domains, including immunology, reproductive biology, and developmental biology, as demonstrated by numerous studies (Khangarot and Das 2010; Boisseaux et al. 2017; Pirger et al. 2018).

Gastropods have an effective internal defense system (IDS) that differs from the immune system of vertebrates. This system comprises both cellular and humoral elements that act together to combat the infection (Saad et al. 2014, 2017; Coustau et al. 2015; Le Clec’h et al. 2016). The hemolymph of freshwater snails contains hemoproteins and different types of phagocytic cells called hemocytes. Previous studies reported that the immune mechanism that influences compatibility between Schistosoma mansoni and B. alexandrina snails depends primarily on the hemocytes (Pila et al. 2017), which play an important role in the recognition and attack of foreign bodies through different processes such as encapsulation, phagocytosis, and releasing some cytotoxic mediators (Bayne 1990; Miller et al. 2001; Cavalcanti et al. 2012).Among the other humoral mediators, they released cytokines in result to immune response, cytokine receptors transduce important signals that regulate proliferation, survival, activation status, and trigger effector functions (Romee et al. 2014). Hemocytes are also directly engaged in pathogen and parasite killing, via the oxidative burst reaction, resulted in a rapid generation of toxic reactive oxygen species (ROS), that arises from a membrane bound enzyme NADH-oxidase (Sokolova 2009).

Examining the influence of Sarcophaga argyrostoma (Robineau-Desvoidy, 1830) larvae hemolymph on snails as a model becomes crucial in understanding the environmentally friendly aspects of its impact. The significance lies in harnessing the immunological potential of natural agents to address issues related to snail immunomodulation, while minimizing ecological impact. Utilizing insect products like S. argyrostoma hemolymph offers a sustainable alternative to synthetic chemicals, mitigating potential adverse effects on non-target organisms and the environment. This approach aligns with eco-friendly methods, emphasizing a holistic and sustainable strategy for disease prevention. By exploring the impact of larvae hemolymph on snails, we gain insights that contribute not only to our understanding of snail biology but also prioritize the preservation of natural habitats and biodiversity, ensuring a balanced and enduring approach. So, using natural agents, such as insect products of the larval hemolymph from S. argyrostoma, can develop new strategies to protect human health and preserve the natural habitats and biodiversity.

Material and methods

Insect colony: to attract gravid females for larviposition, a piece of fresh meat was placed in an open wooden box to start the stock colony of S. argyrostoma flies, the larvae were identified morphologically according to Shaumar and Kawal (1982). Sarcophaga argyrostoma's last larval instars were moved and the hemolymph was extracted from the larvae (60 larvae/1 ml hemolymph) according to Cytorynska et al. (2007). The hemolymph sample was analyzed using GC–MS to determine the chemical composition and the components were identified according to Mohamed (2021).

Snails’ collection: the snails used in the present study were laboratory reared B. alexandrina. The experimental snails were kept individually in 500 ml jars and maintained under constant temperature of 25 ± 2 °C. A photoperiod of 12 h per day was applied, each snail was supplied daily with one bunch of lettuce (Ibrahim et al. 2023a).

Two groups were prepared; snails exposed to larval hemolymph and control group. Three replicates, each of 10 snails/L, were prepared for each group. Observations were recorded for both control and exposed snails (Mohamed and Ishak 1981).

Hemolymph samples of snails were collected after 48 h of larval hemolymph exposure as previously described by Michelson (1966) to undergo different assessment.

TAC and MDA analyses were estimated as oxidative biomarkers using commercial kits (Biodiagnostic Co., Dokki, Giza, Egypt) as described in the manufacturer’s instructions by spectrophotometer.

The production of interferon‐gamma (IFN‐γ; Cat. No. 430801) and interleukin-6 (IL-6; Cat. No.431301) in hemolymph were determined. As they are biomarkers of inflammatory mediators by enzyme linked immunosorbent assay (ELISA), BioLegend, Inc., (ISO 13485 certified; BioLegend Way, San Diego, CA 92121), and the absorbance was measured at 450 nm by UV max ELISA reader (molecular Devices Crop).

Histopathological changes of head foot region and digestive glands of snails were detected. Snails from each treatment and control group were randomly selected; the shell was gently broken and removed carefully. Head foot and digestive gland were carefully separated, fixed in Bouin's solution for 24 h, dehydrated, cleared, embedded in paraffin, sectioned serially, and stained with hematoxylin and eosin (Romeis 1989), mounted, and examined using an Olympus System Microscope with an automatic camera.

The statistical analysis was performed with the aid of the SPSS computer program (version 23.0 windows).

Results

GC- MS results

The hemolymph is a complex biological fluid containing a wide variety of compounds. The results showed that the major chemical compound of larval hemolymph was amino acid (71.11 mg/100 ml) while phenolic content showed the lowest concentration; 1.03 mg/100 ml. Some substances may not be amenable to GC–MS analysis due to their chemical properties and volatility (Table 1).

Table 1 The major chemical compounds of S. argyrostoma larval hemolymph by GC–MS analysis
Full size table

Molluscicidial activity

After 24 h of exposure and another 24 h for recovery, up to 500 ppm of the larval hemolymph showed no toxicity on adult B. alexandrina snails.

Oxidative stress

The oxidative stress was determined by means of malondialdehyde (MDA) and total antioxidant capacity (TAC) concentrations;the data are summarized in Fig. 1. The decrease of MDA concentrations was observed after exposure to hemolymph reaching 31.9 ± 1.48 compared to the control group (42.4 ± 1.6). While the results of TAC in exposed group (43.67 ± 1.32) were significantly (p < 0.05) higher than control (20.4 ± 1.95).

Fig. 1
figure 1

TAC and MDA levels in control and larval hemolymph-exposed snails after 48 h. Values are expressed as mean ± standard deviation (SD), n = 7 snails. MDA malondialdehyde and TAC total antioxidant capacity. *p < 0.05 vs. control group

Full size image

Immunomodulatory activity

IFN-γ and IL-6 concentrations statistically decreased in the group of snails exposed to hemolymph compared to the control after 48h of exposure, with mean reduction values of 44.3 ± 3.01 and 35.3 ± 1.93, with significant decrease (p < 0.05) compared to the control (75.3 ± 2.15 and 66.03 ± 2.29) respectively. The data is expressed in Fig. 2.

Fig. 2
figure 2

IFN-γ and IL-6 (pg/ml) levels from control and larval hemolymph exposed-snails after 48 h. Values are expressed as mean ± standard deviation (SD), n = 7 snails. IFN-γ: interferon-gamma and IL-6: interleukin-6. * p < 0.05 vs. control group

Full size image

Histology

Histological pattern of head foot and digestive gland of B. alexandrina snails is photographed in Fig. 3. The normal head foot region has an outer cuticular layer as a protective layer of the foot. Inner to this lining there is a tall columnar epithelium with basal nuclei in its cell. Amongst the columnar epithelium there are modified sacs like cells in the form of unicellular glands which open through the cuticular layer exterior to the foot surface. These unicellular glands are involved in mucous secretion. Embedded in between, there are transverse muscle fibers and longitudinal muscle fibers. The major part of the foot muscles is made up of thickly arranged oblique muscle fibers (Fig. 3a). The head foot of B. alexandrina snails exposed to S. argyrostoma larval hemolymph showed histopathological changes including degeneration of muscle fibers, empty spaces or vacuoles and accumulation of S. argyrostoma larval hemolymph as seen in (Fig. 3b).

Fig. 3
figure 3

Histological investigations of head foot and digestive gland of B. alexandrina snails 48 h post exposure. a Normal head foot of the control group; connective tissue (CT), mucous cell (MC); muscle layer (ML) and columnar epithelia (CE). b Exposed B. alexandrina snail to S. argyrostoma larval hemolymph; degenerated muscle fiber and empty spaces or vacuoles within muscle (V), atrophy of muscle layer (ML) connective tissues with edema and densely stained in outer layer (TA). c The control group with normal digestive gland having Lumen (L); digestive cell (DC); excretory cell (EC) and connective tissue between hepatopancreatic tubules (CT). d Exposed B. alexandrina snails to S. argyrostoma larval hemolymph with necrotic changes of excretory cells (EC) and digestive cells (DC), dilated lumen and more than two tubules are fused forming one larger lumen (L); connective tissue between hepatopancreatic tubules (CT) and cellular vacuoles were noticed (V)

Full size image

The digestive gland of B. alexandrina, also known as hepatopancreas is a compound tubular gland interspersed with connective tissues, the digestive gland tubules are lined with simple epithelial cells arranged around a narrow irregular lumen and consist of two main cell types, digestive cells (columnar with basal nuclei) and excretory cells (contain granular cytoplasm) with a few mucous cells scattered among them (Fig. 3c). A histological change of digestive gland was observed as a result of treatment of snails with S. argyrostoma larval hemolymph. It was noticed that there was not any difference of digestive gland architectures of all the treated snails in response to hemolymph. However, some tubules were more affected than others within the same digestive gland. The connective tissues between the tubules of digestive gland showed histological changes which included degeneration of all cells. Consequently, the lumen of tubules became large. The rupture of epithelial cells resulted in absence of cell boundaries and diffusion of cellular content through the tissue. The alterations observed in digestive gland of exposed snails were vacuolation of cells, filling the lumen with hyaline substances and appearance of necrotic areas (Fig. 3d).

Discussion

The presence of different pleiotropic cytokines (IL-1, IL-2, IL-6 and IFN-γ-like molecules) in gastropod molluscs has a role in immunoregulation. The central role played by the present cytokines in inflammation and its involvement in pathological processes such as parasitic infection has led us to search for the presence of IL-6 and IFN-γ-like mediators in B. alexandrina snails after exposure to S. argyrostoma larval hemolymph.

In the present study, IFN-γ and IL-6 concentrations were statistically decreased in the snails exposed to larval hemolymph compared to the control, which was due to changes in hemocytes activities to modulate immune response and mediate phagocytic activity, hence the cellular factors including phagocytic cells in the hemolymph can recognize self-material from the non-self-material. These hemocytes circulate within the hemolymph and reside in the connective tissues (Le Clec’h et al. 2022). Additionally, they are assumed to be predominantly responsible for wound repair, which necessitates aggregation at the injury site (Barçante et al. 2012).

IFN- γ reduction in the study may be attributed to the effective modulation followed utilization of larval hemolymph in the maintenance of the immune state of the snails. This change may be attributed to hemocytes activation to exert immune-regulatory and -modulatory activities. IL- 6 also significantly decreased in the present study, which is needed in the function of the immune system, particularly in the mediation of inflammatory response, that is usually triggered and aligned with maintenance of immune status (Mashaal and Sadek 2021; Iwuozo et al. 2022). These findings come in parallel with those of Joung et al. (2014) and Qian et al. (2012) who reported that the exposure of RAW264.7 macrophage cell lines to Haliotis discus hannai Ino, 1953, resulting in inhibition of oxidative mediators, decreasing inflammatory cytokines such as TNF-a, IL-6, and IL-1b and increasing macrophage activity.

The current study exhibited an elevation in total antioxidant capacity, in contrast to malondialdehyde activity which showed statistically significant reduction in the snails exposed to larval hemolymph compared to the control, which was due to the protective mechanisms of mollusc’s including several processes which are involved in the interactive immune response of snails, such as oxidative activity, phagocytosis, lysozymes, and lectin formation (Al-Khalaifah 2022). This finding come in parallel with Farghaly and Sadek (2020) who stated that hemolymph of S. argyrostoma larva treatment significantly inhibited host organ damage at a cellular level due to toxic release, this may be due to activation of antioxidant activity that is related to activation of mollusc’s innate immune system or non-specific immunity against a wide range of different factors. A lot of evidence has proven the existence of immune components that can combat foreign elements (Sadek et al. 2021; Al-Khalaifah et al. 2022; Al-Surrayai and Al-Khalaifah 2022; Al-Khalaifah 2020). In molluscs, there is no adaptive immunity, and they depend on innate and humoral components for immunomodulation. They exhibit a broad array of highly specific innate immune receptors for immune recognition, all without incurring the autoimmune response in the absence of adaptive immunity (Guo and Ford 2016). Their defense line consists mainly of cellular defense; however, humoral factors are essential in modulating and regulating the immune response.

The common features of histopathological effects in snail head-foot are shrinkage in the mucus secreting unicellular glands, atrophy within muscles splitting fiber tissues and several vacuoles (El-Khayat et al. 2015). The present histopathological observations in the head-foot region were degenerated muscle fiber, vacuoles, and atrophy of muscle layer accompanied with connective tissues with edema in treated B. alexandrina. While the induced histopathological changes in the digestive gland under the effect of S. argyrostoma larval hemolymph were cells vacuolation, hyaline substance filled the lumen of the tubules and necrotic focal areas. These findings agree with those recorded by Yousef and EI-Kassas (2013) who observed histopathological effects of three Egyptian wild plant-extracts, as botanic toxic agents; Euphorbia splendens, Ziziphus spina-christi and Ambrosia maritima on digestive gland of infected-target snails, they showed numerous vacuoles in digestive and excretory cells. El-Deeb and El-Nahas (2005) recorded severe damage in the digestive glands of B. alexandrina snails treated with Euphorbia nubica extract. Also, El-Khayat et al. (2018) found that treatment with Anagallis arvensis plant caused cells vacuolation, hyaline substance filled the lumens of the tubules and necrotic focal areas in the digestive gland while, Viburnum tinusca caused vacuolar degeneration with necrotic changes. At this manner S. argyrostoma hemolymph acts as natural and eco-friendly agent which has shown potential for stimulation the immunity of B. alexandrina snails as immunomodulator with anti-inflammatory and antioxidant activities.

The paradoxical histological adverse effects, despite improved antioxidant and anti-inflammatory parameters in the current study, may arise from compound-specific actions. While the hemolymph as a natural compound enhances overall antioxidant and anti-inflammatory responses, it might concurrently induce specific adverse effects on snail histology. This dual role in immune regulation could disrupt certain pathways or cellular processes, leading to histological damage. Metabolic interference is another potential factor, where the natural compound may impact essential metabolic pathways despite its positive effects. To untangle these complexities, further detailed investigations at molecular, cellular, and histopathological levels are recommended to uncover the underlying mechanisms.

Conclusion

In summary, Sarcophaga argyrostoma larval hemolymph as natural agents may contribute to a more sustainable and ecofriendly responsible approach to disease control as antioxidant and anti-inflammatory mediator. By prioritizing both human health and environmental well-being, we can strive for a harmonious coexistence with nature. These findings open avenues for innovative and sustainable therapeutic interventions rooted in natural sources and highlight the potential benefits of S. argyrostoma larval hemolymph in mitigating biological responses. Overall, further studies are needed to determine the effectiveness and environmental impact of hemolymph on the survival rate of infected snails and significance of the snail immunity during mediated schistosomiasis, considering environmental sustainability and host health implications.