Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects, the cell‐mediated immune response involves an active role of hemocytes in phagocytosis, nodulation, and encapsulation. Although these processes have been well documented in multiple species belonging to different insect orders, information concerning the immune response, particularly the hemocyte types and their specific function in the black soldier fly Hermetia illucens, is still limited. This is a serious gap in knowledge given the high economic relevance of H. illucens larvae in waste management strategies and considering that the saprophagous feeding habits of this dipteran species have likely shaped its immune system to efficiently respond to infections. The present study represents the first detailed characterization of black soldier fly hemocytes and provides new insights into the cell‐mediated immune response of this insect. In particular, in addition to prohemocytes, we identified five hemocyte types that mount the immune response in the larva, and analyzed their behavior, role, and morphofunctional changes in response to bacterial infection and injection of chromatographic beads. Our results demonstrate that the circulating phagocytes in black soldier fly larvae are plasmatocytes. These cells also take part in nodulation and encapsulation with granulocytes and lamellocyte‐like cells, developing a starting core for nodule/capsule formation to remove/encapsulate large bacterial aggregates/pathogens from the hemolymph, respectively. These processes are supported by the release of melanin precursors from crystal cells and likely by mobilizing nutrient reserves in newly circulating adipohemocytes, which could thus trophically support other hemocytes during the immune response. Finally, the regulation of the cell‐mediated immune response by eicosanoids was investigated.


Introduction
In insects, cellular and humoral components of the immune system are rapidly triggered by different nonself agents to counteract infections (Eleftherianos et al., 2021a). Although the distinction between cellular and humoral immune reactions as two different categories is not straightforward, hemocytes represent key players in almost all the processes by which insects clear foreign agents from their body. In fact, these circulating cells are not only directly involved in the mechanisms underlying the cellular response, such as phagocytosis, nodulation, and encapsulation (Strand, 2008;Eleftherianos et al., 2021a); they are largely responsible for producing most of the humoral components, too. For example, intermediates of the prophenoloxidase (proPO) cascade, lysozyme, and antimicrobial peptides are synthesized by both the fat body and hemocytes (Lemaitre & Hoffmann, 2007;Eleftherianos et al., 2021b).
Considerable effort has been invested in studying and classifying hemocyte populations in Diptera (Lanot et al., 2001;Pal & Kumar, 2014), Lepidoptera (Tan et al., 2013;Boguś et al., 2018), Coleoptera (Kwon et al., 2014), Orthoptera (Cho & Cho, 2019), Hemiptera (Schmitz et al., 2012), and Hymenoptera (Gábor et al., 2020). In these studies, multiple types of hemocytes were identified, each with a specific role, and significant differences were demonstrated not only among insect orders, but also among genera that belong to the same order (Hillyer & Christensen, 2002;Nakahara et al., 2009;Gábor et al., 2020). This complex scenario is mostly the result of the multitude of morphological, histochemical, and molecular markers that have been used to study these immune cells over the years (Eleftherianos et al., 2021a). Hence, the nomenclature and terminology used to designate hemocytes vary among insect species and differ from order to order and species to species. For example, while granulocytes, plasmatocytes, spherulocytes, and oenocytoids are the cells most widely described in Lepidoptera (Nakahara et al., 2009), plasmatocytes, crystal cells, and lamellocytes, but not granulocytes, were reported in the dipteran Drosophila melanogaster (Brehélin, 1982). A comparison with other Diptera, such as Aedes aegypti and Culex quinquefasciatus, which share the presence of oenocytoids, and Glossina morsitans, in which they are absent (Kaaya & Ratcliffe, 1982), clearly demonstrates that species of this order do not share a general hemocyte pattern. A further level of complexity arises if we consider that different hemocyte types can perform the same function in different insect orders, such as for crystal cells of D. melanogaster and oenocytoids of Lepidoptera, which both produce proPO (Ribeiro & Brehélin, 2006).
The rapid activation of hemocytes by different nonself agents (Hillyer et al., 2003a;Manachini et al., 2011) promotes proliferation and activation of phagocytosis and/or encapsulation processes that last for a long time (Hillyer et al., 2003a;Ling & Yu, 2006;Hwang et al., 2015), indicating that the cellular component of the immune system is maintained active until the threat is completely brought under control. However, as mentioned above, hemocytes also intervene in the production of several humoral molecules. Larvae of the dipteran Hermetia illucens infected with bacteria represent an interesting example, where 3 h are sufficient to start transcription of antimicrobial peptide-coding genes in hemocytes, whereas remarkable transcription levels can be detected only later (6-14 h) in the fat body (Bruno et al., 2021), confirming the key role of hemocytes to quickly counteract bacterial cell proliferation.
In insects, the rapid and coordinated activation of cellular and humoral processes is mediated by the diffusion of various signaling molecules produced upon bacterial challenge (Kim et al., 2018). Among them, eicosanoids act as ultimate downstream mediators in many physiological processes in insects, including activation of the cellular immune response (Stanley, 2006;Stanley & Kim, 2014). Eicosanoids are oxidized derivatives of 20-carbon polyunsaturated fatty acids, most frequently arachidonic acid (C20:4n-6) metabolites (Kim et al., 2018). They include prostaglandins, thromboxanes, leukotrienes, and lipoxins biosynthesized by enzymatic oxygenation reactions of these fatty acids. In insects, hemocytes and the fat body produce eicosanoids, frequently through the hydrolysis of phospholipids by phospholipase A2 (PLA2). These metabolites are fundamental in activating cellmediated processes such as phagocytosis, nodulation, and encapsulation in different insect orders (Stanley, 2006;Stanley & Kim, 2014).
Due to the marked differences in hemocyte pattern and behavior, it is mandatory to expand our knowledge of immune cells in insects and investigate how they respond to different immune challenges, especially in those species that are increasingly being used for their relevant economic importance Tettamanti et al., 2022). In this respect, the larvae of the black soldier fly (BSF), H. illucens, are attracting growing interest as they can be reared on different organic waste and the insect biomass can then become a valuable source of protein and lipids for feed formulation (Hawkey et al., 2021), bioplastic manufacturing (Nuvoli et al., 2021), and biodiesel production (Jung et al., 2022). The ability of these larvae to grow on decaying materials, which are potentially rich in pathogens, has likely contributed to shape their immune system to efficiently respond to infections. However, studies of the immune response of this insect are limited (Vogel et al., 2018;Bruno et al., 2021;Vogel et al., 2022) and very little is known about the morphological and functional features of its hemocytes (Zdybicka-Barabas et al., 2017;von Bredow et al., 2021).
To expand knowledge on the immune response of BSF larvae, herein we assessed if (i) certain hemocyte types are key players in specific cellular responses; (ii) the percentage and dynamics of hemocyte population change in response to infection; and (iii) eicosanoids mediate cellular immune interactions. To evaluate these aspects, we performed an in-depth characterization of hemocytes in H. illucens larvae and explored the roles they play in response to immune challenge; we also examined the involvement of eicosanoids in the modulation of hemocyte populations in response to immune challenge.

Insect rearing
Insects were reared according to Pimentel et al. (2017). Briefly, after hatching, larvae were placed in a humid chamber and fed on standard diet for Diptera (50% wheat bran, 30% corn meal, and 20% alfalfa meal mixed at a 1 : 1 ratio dry matter : water) (Hogsette, 1992). After 4 days, batches of 300 larvae were placed in 16 × 16 × 9-cm plastic containers with ventilated lid, fed on the same diet, and kept in the dark at 27 ± 0.5°C and 70% ± .5% relative humidity. Once at the pupal stage, insects were removed from the rearing substrate and transferred to a net cage until adult eclosion. Flies were maintained at 30 ± 0.5°C, 70% ± 5% relative humidity, with a 12 : 12 h light : dark photoperiod and reared as brood stock (Bruno et al., 2019a).
B-Agarose and DEAE-Sephadex A-25 beads (Merck) were selected for analyzing encapsulation (Bruno et al., 2021). Before injection into the larvae, beads were washed and resuspended in sterile PBS, approximately 5 beads/μL.

Injection of larvae and hemolymph collection
Last instar larvae were washed with tap water to remove the rearing substrate from the cuticle and then with 0.5% sodium hypochlorite (in tap water, volume/volume [v/v]) and 70% ethanol (in distilled water, v/v). Insects were injected with: (i) 5 μL of 1 × 10 5 CFU/mL E. coli/M. luteus mix with a Hamilton 700 10-μL syringe (Hamilton, Reno, NV, USA) or (ii) 5 μL of B-Agarose or DEAE-Sephadex A-25 beads (Merck) with a sterile 1-mL disposable syringe. Injections were performed between the third last and penultimate metamere of the larva. To avoid infections caused by bacteria present in the diet, injected larvae were kept under sterile conditions at 27 ± 0.5°C and 70% ± .5% relative humidity, in the absence of feeding substrate. As the aim of the present study was to analyze the cell-mediated response triggered by the entrance of bacteria through lesions on the body surface, uninjected larvae (naïve) were used as controls, as reported previously (Bruno et al., 2021). To exclude unwanted side effects caused by needle puncturing or PBS injection, specific control experiments were conceived. In detail, total hemocyte count, as well as quantification of adipohemocytes, crystal cells, and granulocytes, were performed by using the hemolymph collected from larvae subjected to puncture with a sterile needle or injected with 5 μL of sterile PBS. Results confirmed a significant difference between control groups and infected larvae (Supporting Information Fig. S1).
Larvae were analyzed from 5 min to 24 h after injection of bacteria or beads (see Results for details). They were rapidly cooled down on ice for 30 s and the hemolymph was collected by cutting the cephalic region with sterile scissors and gently squeezing the larva. The number of hemocytes collected from these larvae was compared with that from larvae kept at room temperature to exclude that the exposure of insects to low temperatures for a few seconds could affect the amount of immune cells (1590 ± 80 hemocytes/μL at low temperature and 1567 ± 53 hemocytes/μL at room temperature-the experiments were performed in triplicate).
The hemolymph was collected in plastic tubes on ice to avoid proPO activation. About 20 μL of hemolymph was collected from each larva. Each experiment was performed in triplicate by pooling samples of hemolymph collected from at least 15 larvae; for transmission electron microscopy (TEM), flow cytometry, quantification of prostaglandin E 2 (PGE 2 ), and analysis of PLA2 activity, hemolymph was collected from at least 50 larvae.

Hemocyte characterization and quantification
Hemocyte counts After collection, hemolymph was diluted 1 : 10 with 0.4% Trypan blue (ThermoFisher, Waltham, MA, USA) and loaded into FAST READ 102 counting chambers (Biosigma S.R.L., Cona, Italy). The total number of hemocytes in control larvae and larvae injected with bacteria was calculated according to the manufacturer's instructions.

Giemsa staining and differential hemocyte counts
A 200-μL sample of hemolymph was poured on sterile round glass coverslips, kept in the dark for 15 min to allow the adhesion of hemocytes to the glass, and then cells were fixed with 5% formalin in PBS. Coverslips were air-dried and incubated for 10 min using the MGG quick stain kit (Bio-Optica, Milano, Italy) (diluted 1 : 20 in sterile PBS). After three washes with PBS, coverslips were mounted on microscope slides with Eukitt (Bio-Optica) and analyzed with an Eclipse Ni-U microscope (Nikon, Tokyo, Japan) equipped with a DS-SM-L1 digital camera (Nikon).
Differential hemocyte counts were performed by using hemolymph of control larvae and larvae injected with bacteria or chromatographic beads, after Giemsa staining. Cells from 10 randomly selected fields (50× magnification) were counted. At least 20 cells for each cell type were considered for size determination. Differential hemocyte counts were expressed as the percentage of each hemocyte type from the total number of cells counted (Silva et al., 2002). Each experiment was performed in triplicate. (Corcoran & Brückner, 2020). For this purpose, naïve larvae and larvae injected with bacteria or chromatographic beads were placed in 2-mL plastic tubes with sterile PBS and then heated at 65°C for 22 min in a Thermomixer comfort (Eppendorf, Hamburg, Germany) (Corcoran & Brückner, 2020). Hemolymph was then collected, diluted (2 : 1 v/v) with Schneider's Insect Medium (Merck), and placed in a 96-well plate. Cells undergoing melanization were identified by using an inverted Olympus IX51 micro-scope (Olympus, Tokyo, Japan) equipped with a C-P20M camera (Optika Microscopes Italia, Ponteranica, Italy).

Identification and quantification of adipohemocytes
Adipohemocytes were identified and quantified by using an osmium staining protocol adapted from Belazi et al. (2009). Hemocytes were isolated from naïve larvae and larvae injected with bacteria or chromatographic beads, poured on sterile round glass coverslips as described in "Giemsa staining and differential hemocyte counts" to allow adherence, and then fixed with 5% formalin in distilled water (v/v) for 10 min in the dark. Coverslips were washed with PBS and then incubated with 1% osmium tetroxide in PBS (v/v) for 1 min in the dark. They were repeatedly washed with distilled water and dehydrated with an ascending ethanol series. Finally, coverslips were mounted on microscope glass slides with Eukitt (Bio-Optica) and analyzed with an Eclipse Ni-U microscope (Nikon) equipped with a DS-SM-L1 digital camera (Nikon). Adipohemocytes were quantified by analyzing 10 different images (50× magnification) randomly selected from each independent experiment. Each experiment was performed in triplicate.
Transmission electron microscopy Hemocytes were fixed by mixing 1 mL of hemolymph with 2% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (1 : 1, v/v), pH 7.4, overnight at 4°C. After centrifuging at 200 × g for 10 min, cell pellets were postfixed in 2% osmium tetroxide in 0.1 mol/L sodium cacodylate buffer for 20 min at room temperature in the dark. Samples were then dehydrated in an ascending ethanol series and embedded in an Epon-Araldite 812 mixture. Ultrathin sections (70 nm thick) were obtained with Leica Reichert Ultracut S (Leica, Wetzlar, Germany) and stained with lead citrate and uranyl citrate. Specimens were observed with a JEM-1010 TEM (Jeol, Tokyo, Japan) equipped with a Morada digital camera (Olympus)-Centro Grandi Attrezzature, University of Insubria.
Immunocytochemistry After adhesion on sterile round glass coverslips as described in "Giemsa staining and differential hemocyte counts," hemocytes were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Then, coverslips were washed with PBS. Preincubation with blocking solution (2% bovine serum albumin [BSA] with 0.01% Tween-20 in PBS) for 30 min preceded incubation with a rabbit anti-phospho-Histone 3 antibody ([#06-570, Merck], dilution 1 : 500 in 2% PBS/BSA [v/w] with 0.01% Tween-20) for 1 h at room temperature. This antibody is able to recognize mitotic cells in H. illucens (Bonelli et al., 2019). After several washes with PBS, cells were incubated with an anti-rabbit Cy3-conjugated secondary antibody (dilution 1 : 300 in 2% PBS/BSA [v/w] with 0.01% Tween-20) (Abcam, Cambridge, UK) for 1 h at room temperature. After several washes with PBS, hemocytes were incubated with DAPI (100 ng/mL in PBS) for 5 min to stain nuclei and then mounted with Citifluor (Citifluor Ltd, London, UK). Specimens were analyzed with an Eclipse Ni-U microscope (Nikon) equipped with a DS-SM-L1 digital camera (Nikon). Phospho-histone 3-positive cells were quantified by analyzing 10 different images (50× magnification) randomly selected from each independent experiment. The experiments were conducted in triplicate. Negative controls were performed by omitting the primary antibody.

Flow cytometry analysis of apoptotic cells
Apoptotic cells in the hemolymph of naïve and infected larvae were quantified by flow cytometry. Here, 1 mL of hemolymph was collected from 50 larvae 30 min, 1 h, and 2 h after injecting the bacteria. Samples were then centrifuged at 200 × g for 10 min. The pellets were washed three times with sterile PBS, fixed in cold 70% ethanol, and stored at −20°C until use. For FACS analysis, cells were centrifuged for 10 min at 1100 × g and DNA was stained with 250 μL of 50 μg/mL propidium iodide in PBS in the presence of 30 U/mL of RNAse A. Samples were analyzed with a FACSCalibur flow cytometer and data were processed using CellQuestPRO software (Becton Dickinson, Franklin Lakes, NJ, USA). Fluorescence emission of propidium iodide was collected through a 575-nm band-pass filter, acquired in logarithmic mode, and the percentage of apoptotic cells was determined based on sub-G1 peaks detected in monoparametric histograms (Riccardi & Nicoletti, 2006). The analyses were conducted in triplicate for each time-point analyzed.
PLA2 activity and PGE 2 quantification Activity of PLA2 and quantification of PGE 2 were assessed in both cell-free and total hemolymph samples collected from uninfected and infected larvae at 30 min and 3 h after infection. After isolation, hemolymph samples were diluted with Schneider's Insect Medium (Merck) (4 : 1 ratio) and a few crystals of N-phenylthiourea (Sigma-Aldrich, St Louis, MO, USA) were added to the solution to avoid proPO activation. Cell-free hemolymph samples were obtained by centrifuging the hemolymph at 10 000 × g for 10 min at 4°C. However, total hemolymph samples were first sonicated for 3 min for cell lysis and then centrifuged at 10 000 × g for 10 min at 4°C. Supernatants were collected and stored at −80°C until use.
The PLA2 activity was detected by using the fluorometric EnzChek Phospholipase A2 Assay Kit (Invitrogen, Carlsbad, CA, USA), which provides sensitive and rapid real-time monitoring of PLA2 activity. Briefly, the hemolymph samples were diluted 1 : 5 with the reaction buffer to ensure that conditions were appropriate for the enzymatic reaction. Measurements were performed using the fluorescence endpoint method (excitation: 485/20 nm, emission: 528/20 nm) with a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). Quantitative eicosanoid analysis was carried out using the Human Prostaglandin E 2 Elisa Kit (Bioassay Technology Laboratory, Shanghai, China). Absorbance (wavelengths: 450 nm) was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek). PLA2 activity and PGE 2 concentration were assessed by means of their respective standard curves. Each test was performed in three independent replicates according to the manufacturer's instructions.

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 7.00 (GraphPad software, La Jolla, CA, USA). Quantification of apoptotic cells and PGE 2 over time, PLA2 activity, as well as the variation in numbers of adipohemocytes, granulocytes, and crystal cells under different conditions (i.e., control larvae and larvae injected with the bacterial mix or chromatographic beads) were analyzed using one-way analysis of variance followed by Tukey's multiple-comparison post hoc test. Hemocyte counts and mitotic hemocytes were analyzed using the unpaired Student's t-test. The normality of all the data was checked and confirmed with the Shapiro-Wilk test. Differences between groups were considered statistically significant at a P value less than 0.05.

Morphological characterization of hemocytes
Five different types of hemocytes were identified in naïve larvae according to their morphology and appearance after Giemsa staining, namely: prohemocytes, plasmatocytes, lamellocyte-like cells, crystal cells, and granulocytes ( Fig. 1A-E). As reported in Table 1, plasmatocytes represented the most abundant population, corresponding to 91% of circulating cells in the hemolymph. A smaller fraction of hemocytes (about 7%) was represented by prohemocytes, whereas the remaining cell types amounted to less than 1% each. Immature  Ultrastructural analysis showed that prohemocytes were round or oval cells with a high nucleus : cytoplasm ratio ( Fig. 2A, B). A thin, homogeneous layer of cytoplasm almost devoid of rough endoplasmic reticulum (RER) and mitochondria surrounded the centrally located nucleus (Fig. 2B). Plasmatocytes were, in contrast, rich in organelles, as a well-developed RER, numerous mitochondria, and lysosomes (Fig. 2C, D). These cells showed a lobed, eccentrically located nucleus as well as numerous membrane protrusions (Fig. 2C). Lamellocytelike cells were elongated in shape with tapered ends, with a large oval nucleus and a developed RER (Fig. 2E, F).
Crystal cells were the largest hemocytes in naïve larvae (Fig. 2G, H) (Table 1). They were characterized by numerous, large crystalline inclusions in the cytoplasm that could extend throughout the cell (Fig. 2H), a large, round nucleus, and a smooth membrane without protrusions (Fig. 2G, H). Finally, granulocytes were spherical in shape (Fig. 2I). They were characterized by an irregular membrane, a centrally located, lobed nucleus, and spherical or ovoidal electron-dense granules in the cytoplasm (Fig. 2I, J). The dimensions of all the hemocyte types observed in naïve larvae are reported in Table 1. Interestingly, a sixth cell type, i.e., adipohemocytes, was detected only in larvae subjected to immune challenge (Figs. 1F and 2K, L). These round or oval cells were large (16 ± 1.2 μm; n = 10), with a round or slightly   (Fig. 2K). Their membrane was irregular and characterized by thin protrusions. Numerous lipid droplets and vacuoles were present in the cytoplasm (Fig. 2L).

Activation of hemocytes by immune challenge: phagocytosis, nodulation, and encapsulation
Hemocyte-mediated processes were evaluated by challenging larvae with bacteria and different types of beads ( Figs. 3 and 4).
Phagocytosis was analyzed within a short time after infection, i.e., within 1 h after injection of bacteria (Bruno et al., 2021). Morphological changes in hemocytes confirmed the rapid activation of this cell-mediated process. In particular, numerous membrane protrusions (Fig. 3A) as well as engulfed bacteria (Fig. 3B, E, F) were frequently observed in activated plasmatocytes, from 5 to 15 min after infection. Moreover, phagolysosomes containing degraded bacteria were detected in their cytoplasm within 30 min (Fig. 3C, G). Interestingly, phagocytosis was accompanied by a rapid removal of exhausted hemocytes. In particular, apoptotic cells, clearly recognizable for cytoplasm vacuolization and chromatin condensation (Fig. 3D, H), could be observed as freely circulating cells (Fig. 3D) or engulfed by phagocytes (Fig. 3H) 1 h after infection. These results were confirmed by FACS analysis. In detail, apoptotic hemocytes detected 1 h after infection were significantly more abundant than in naïve larvae at all the time-points (Fig. 5A).
Large bacterial aggregates that could not be phagocytosed soon after infection (10 min) were removed by nodulation (Fig. 3I), as confirmed by plasmatocytes surrounding clusters of bacteria through pseudopodia and melanin presence within the nodule (Fig. 3J).
To analyze encapsulation, neutrally charged B-Agarose beads and positively charged DEAE-Sephadex beads were injected into the larvae and examined 24 h later. At this time-point, these beads are encapsulated to a different extent in BSF larvae (the former are only partially surrounded by hemocytes, whereas the latter are fully encapsulated and melanized) (Bruno et al., 2021). Hence, this approach allowed us to investigate which hemocyte populations were involved in encapsulating the two nonself macroagents. Plasmatocytes, granulocytes, and presumably lamellocyte-like cells were involved in forming a compact cell capsule surrounding DEAE-Sephadex beads (Fig. 4A, B) with a typical arrangement: plasmatocytes and lamellocyte-like cells were localized in the inner part of the capsule, adherent to the foreign body (Fig. 4A, C), whereas granulocytes were distributed in the outer region (Fig. 4B, D). Moreover, encapsulated beads became completely melanized (Fig. 4E). In contrast, just a few hemocytes surrounded B-Agarose beads (Fig. 4G). As for the DEAE-Sephadex beads, plasmatocytes and granulocytes participated in encapsulation (Fig. 4G, H), but neither a fully formed cell capsule nor melanization was observed.

Morphofunctional features of activated hemocytes
To evaluate a potential turnover of exhausted hemocytes removed from hemolymph following phagocytosis (Fig. 3D, H), we compared the numbers of hemocytes in naïve and infected larvae over time. As shown in Fig. 5B, a striking reduction in hemocytes was recorded 30 min after infection (1.59 × 10 6 ± 0.08 × 10 6 cells/mL in naïve larvae and 6.58 × 10 5 ± 0.82 × 10 5 cells/mL in infected larvae). This decrease in cell number was maintained up to 1 h (1.82 × 10 6 ± 0.15 × 10 6 cells/mL in naïve larvae and 7.98 × 10 5 ± 0.88 × 10 5 cells/mL in infected larvae), whereas 3 h after infection, the number of circulating cells was comparable to controls at the same time-point (1.31 × 10 6 ± 0.04 × 10 6 cells/mL in naïve larvae and 1.17 × 10 6 ± 0.05 × 10 6 cells/mL in infected larvae), indicating a recovery in the number  immunostaining demonstrated that this phenomenon was mainly due to cell proliferation, as the percentage of mitotic hemocytes doubled (6.48% ± 0.9%) compared with naïve larvae (3.16% ± 0.2%) 2 h after infection ( Fig. 5C; Supporting Information Fig. S2). Moreover, at the same time-point, the number of apoptotic cells decreased to levels comparable to naïve larvae (Fig. 5A). Collectively, these results showed a strong reduction in apoptosis and the recovery of total hemocyte numbers through cell proliferation 2 h after infection, confirming a turnover of the immune cells.
We also observed significant modifications in the pattern and behavior of some hemocyte populations after the immune challenge. First, adipohemocytes started to circulate upon the entrance of foreign agents in the hemocoel. The presence of these cells in the hemolymph was demonstrated by TEM analysis (Fig. 2K) and osmium staining ( Fig. 6B-D). In particular, adipohemocytes, which were never observed in naïve larvae (Fig. 6A), amounted to 7% ± 0.8% of total hemocytes after infection with bacteria and 9.2% ± 0.5% and 6.5% ± 1.1% after injection of B-Agarose and DEAE-Sephadex beads, respectively (Fig. 6E). Second, crystal cells underwent a change in morphology and number after activation. TEM analysis showed a strong reduction in the amount of cytoplasmic crystals upon bacterial infection (Fig. 7B) or injection with DEAE-Sephadex beads (Fig. 7D) compared with naïve larvae (Fig. 7A) and larvae injected with B-Agarose beads (Fig. 7C). Larval heating triggered the phenoloxidase system such that we could monitor the crystal cell response under different conditions. These experiments confirmed that crystal cells from naïve and B-Agarose bead-injected larvae were black in color because of the presence of crystals in the cytoplasm that, after activation by heat treatment, led to spontaneous melanization of the cell (Fig. 7E, G), whereas the number of crystals in cells isolated from larvae injected with bacteria or DEAE-Sephadex beads (Fig. 7F, H) decreased. Moreover, while crystal cells increased from 0.38% ± 0.24% in naïve larvae to 1.33% ± 0.17% and 1.83% ± 0.15% upon injection of B-Agarose and DEAE-Sephadex beads, respectively, the number of crystal cells remained unchanged after bacterial infection (Fig. 7I). Third, the number of granulocytes increased after injecting bacteria (3-fold compared with naïve larvae) and DEAE-Sephadex or B-Agarose beads (6-fold compared with naïve larvae) (Fig. 8E). TEM analysis of these cells showed an increase in cytoplasmic granules after activation (Fig. 8B-D) compared with controls (Fig. 8A).

PLA2 activity and PGE 2 quantification
Activity of PLA2 was measured 30 min and 3 h after bacterial infection (Fig. 9A). In total hemolymph, the activity of PLA2 remained at basal levels 30 min after the infection (0.28 ± 0.01 U/mL versus 0.24 ± 0.03 U/mL in naïve larvae), whereas the enzyme was strongly activated at 3 h (0.5 ± 0.03 U/mL). No activity was recorded in cell-free hemolymph.
The PGE 2 concentration differed significantly between cell-free and total hemolymph (Fig. 9B). In addition, although the amount of prostaglandin remained constant in cell-free hemolymph, the concentration in total hemolymph increased significantly over time after the infection. Specifically, the amount of this eicosanoid increased from 30.9 ± 4.7 ng/L in naïve larvae to 50.9 ± 1.3 ng/L and to 67.2 ± 4.7 ng/L in infected larvae at 30 min and 3 h, respectively.

Discussion
In the present study, we morphofunctionally characterized the hemocyte populations in larvae of H. illucens, a saprophagous insect that is increasingly being used for waste bioconversion. This work also expands the current knowledge on immune cells in insects in general and confirms the high variability of hemocytes in Diptera. Indeed, except for prohemocytes and plasmatocytes, which are widely conserved among insects, two specific hemocyte populations, granulocytes and adipohemocytes, which have been previously described in the larvae of other Cyclorrhapha such as Sarcophaga ruficornis, Chrysomya megacephala, and Musca domestica, mount the immune response in BSF larvae (Pal & Kumar, 2014). However, no crystal cells were described in any of these Diptera, unlike D. melanogaster larvae (Meister & Lagueux, 2003). Hence, the profile of hemocytes in H. illucens seems to be unique and mixed, which is relevant for the vast majority of cyclorrhaphan Diptera, i.e., prohemocytes, plasmatocytes, granulocytes, and different time-points. (C) Percentage of mitotic hemocytes in naïve (Naïve) and infected (E. coli/M. luteus) larvae at different timepoints. Values represent mean ± SEM. Asterisks represent statistically significant differences between naïve larvae (Naïve) compared with larvae injected with bacteria (E. coli/M. luteus) at different time-points-unpaired Student's t test: (B) for 30 min, t = 8.226, df = 4, P = 0.0012; for 1 h, t = 5.588, df = 4, P = 0.005; for 3 h, t = 0.574, df = 4, P = 0.597; (C) for 1 h, t = 2.06, df = 4, P = 0.109; for 2 h, t = 3.257, df = 4, P = 0.031).   adipohemocytes (Kaaya & Ratcliffe, 1982;Silva et al., 2002;Pal & Kumar, 2014;Dorrah et al., 2019). It differs, however, in the presence of the crystal cells, analogous to that of Drosophila (Meister & Lagueux, 2003), and in the complete absence of spherulocytes and the enigmatic oenocytoids relevant for other Diptera such as mosquitoes (Castillo et al., 2006;Araújo et al., 2008).
We showed that the number of circulating hemocytes in BSF larvae undergoes a marked reduction within 1 h after infection, as in other insects (Kaaya et al., 1986;Li et al., 2019). TEM analysis suggests that this is probably due to the removal of exhausted hemocytes by circulating phagocytic cells, as hypothesized for Bombyx mori (Li et al., 2019). The total number of hemocytes increases again 2 h later, reaching levels comparable to those in control larvae, providing new immune cells to better cope with the infection. This phenomenon can be explained by one of the following mechanisms, or a combination thereof: (i) proliferation of circulating hemocytes, as indicated by H3P immunostaining; (ii) mobilization, following infection, of specific hemocytes (i.e. adipohemocytes) which under healthy conditions adhere to tissues or organs (Hillyer & Christensen, 2002); and (iii) ex novo production by hematopoietic organs of prohemocytes, which generate mature hemocytes (Jung et al., 2005).
We previously showed that hemocytes already circulating in healthy BSF larvae induce phagocytosis, nodulation, and encapsulation as soon as the infection begins (Bruno et al., 2021). As demonstrated in this study, phagocytosis is triggered within a few minutes. Although the kinetics is similar to that in other Diptera (Hillyer et al., 2003a), ultrastructural analysis indicates that only plasmatocytes participate in this process in BSF. This peculiar feature is shared by H. illucens and D. melanogaster larvae (Meister & Lagueux, 2003), but it differs from other Cyclorrhapha, where phagocytosis is mediated by both plasmatocytes and granulocytes (Franchini et al., 1996) or preferentially by the latter (Faraldo & Lello, 2003), and Nematocera (i.e. mosquitoes) in which granulocytes engulf bacteria (Hillyer et al., 2003a;Hillyer et al., 2003b;Castillo et al., 2006;Smith et al., 2016). When we extend the comparison to other holometabolous insects, plasmatocytes have never been described, to the best of our knowledge, as the only phagocytic hemocytes. In many lepidopteran species (i.e. Heliothis armigera, Galleria mellonella, Spodoptera littoralis, and B. mori), for example, the major phagocytic activity has been attributed to granulocytes (Salama & Sharaby, 1985;Tojo et al., 2000;Costa et al., 2005;Wu et al., 2015), whereas in Coleoptera, both oenocytoids and granulocytes act as phagocytes (Giulianini et al., 2003). Hence, it seems that H. illucens larvae, where different populations of hemocytes developed, experienced a strong functional specialization during evolution, at least for phagocytosis.
In contrast to phagocytosis, the type and role of hemocytes involved in encapsulating large foreign agents are highly conserved among insects (Lavine & Strand, 2002). Generally, granulocytes intervene first and release granules to chemoattract plasmatocytes toward the foreign agent (Pech & Strand, 1996), which is progressively surrounded by these cells in a multilayered capsule that can eventually be melanized (Gorman et al., 1998). The cell pattern observed in BSF during encapsulation is comparable to that reported in other insects and, although we were not able to definitely distinguish plasmatocytes from lamellocytes, our results suggest that, in addition to plasmatocytes, also lamellocyte-like cells are likely involved in capsule formation, as demonstrated in D. melanogaster larvae (Rizki & Rizki, 1992). The presence of granulocytes close to both types of beads indicates their active involvement in encapsulation. Moreover, the high number of circulating granulocytes when encapsulation has already been completed (24 h after bead injection), as well as the large number of granules in their cytoplasm, suggest that these cells could help to mount a faster immune response in the case of repeated infections. The increase in granules after injecting both types of beads and the melanization of DEAE-Sephadex beads only indicate that granulocytes are primarily involved in recruiting hemocytes that participate in capsule formation and not in the production of proPO, as in G. mellonella (Schmit et al., 1977). In H. illucens, this zymogen can instead be synthesized by crystal cells (Paro & Imler, 2016), as demonstrated by the large cytoplasmic crystalline inclusions containing enzymes for melanization (Meister, 2004) and by heating assays. These results also demonstrate that, differently from Drosophila (Bidla et al., 2007), crystal cell disruption does not occur in BSF as crystals are likely dissolved progressively in these larvae and their content is released into the hemolymph. In addition, the increase in crystal cells involved in encapsulation over time can support the melanization of nonself agents, as in parasitized D. melanogaster larvae (Sorrentino et al., 2002). Crystal cells likely intervene in nodulation, too, whereby melanin is deposited in the nodule formed by plasmatocytes (Satyavathi et al., 2014). In this case, the process could be triggered by granulocytes, which significantly increase in number upon bacterial infection by releasing their granules and favoring agglutination of bacteria, thus recruiting plasmatocytes and crystal cells (Dean et al., 2004).
As mentioned above, adipohemocytes, which adhere to inner tissues/organs in naïve larvae (Hillyer & Christensen, 2002), start circulating in the hemolymph following infection. According to this change in behavior and the high lipid content in the cytoplasm (Hwang et al., 2015), we hypothesize that these cells play a trophic role toward other hemocytes engaged in the immune response. This function seems to be independent of the stimulus because no difference in their number was observed after different immune challenges.
To summarize, the cellular response in BSF larvae mediated by hemocytes is finely regulated to rapidly counteract the diffusion of foreign agents. Our results demonstrate that phagocytosis is carried out by plasmatocytes only. Granulocytes are instead involved in encapsulation and nodulation, developing a starting core for capsule/nodule formation to isolate/remove large nonself agents/bacterial aggregates from the hemolymph, respectively. Crystal cells participate in these cell-mediated processes by releasing crystals that contain proPO for melanin production. Finally, adipohemocytes likely support trophically other hemocytes during the immune response (Fig. 10).
In insects, the rapid synthesis of prostaglandins, triggered by the activation of PLA2 (Kim et al., 2018), activates hemocytes, phagocytosis, and the proPO system (Mandato et al., 1997;Roy & Kim, 2021). In particular, PGE 2 can mediate the occurrence of apoptosis in G. mellonella hemocytes (Wrońska et al., 2022), hemocyte spreading (Kim et al., 2020), and the release of proPO from oenocytoids in Spodoptera exigua (Shrestha et al., 2011). Our results for PLA2 and PGE 2 indicate that eicosanoids are potentially involved as mediators of the cellular immune responses of BSF larvae against bacterial infections. This hypothesis is corroborated by the active role of hemocytes in the synthesis of prostaglandins, as shown by comparing cell-free and total hemolymph samples. Future studies are needed to dissect how PGE 2 regulates specific cell-mediated immune mechanisms in this insect.
Although recent studies aimed to identify hemocyte populations in BSF larvae, they underestimated the number, variability, and behavior of circulating hemocytes in this insect (Zdybicka-Barabas et al., 2017;von Bredow et al., 2021). Thanks to TEM analysis, we identified specific cell populations-some of which were not properly classified or were completely overlooked in previous studies-and defined ultrastructural features of hemocytes that have been neglected so far (Zdybicka-Barabas et al., 2017;von Bredow et al., 2021). For example, although von Bredow et al. (2021) described granules in the cytoplasm of specific cells, these were not identified as granulocytes; moreover, light microscopy analysis performed in that study did not allow identification of lamellocyte-like cells and adipohemocytes. Most importantly, our ultrastructural analysis was fundamental to unravel the morphofunctional modifications of hemocytes upon different immune challenges and the potential role of these cells. Although the use of specific cellular and molecular markers is mandatory to further characterize BSF hemocytes, this study, together with an in-depth knowledge of the humoral components (Bruno et al., 2021) and of the role of the midgut as a protective organ against infections (Bonelli et al., 2019;Bruno et al., 2019b), will provide us with a better understanding of the physiological mechanisms that help BSF larvae to survive within rearing substrates that are extremely rich in microbes, which would be harmful for most insect species.