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Live-cell images of cricket hemocytes infected with E. coli or Sephadex beads. (A) Light microscope images showing hemocytes cultured with E. coli. As the incubation time increased, nets (amoeba-like hairs or extracellular traps; indicated by black arrows) were produced by specific hemocytes. All six types of hemocytes were aggregated into large clusters by these nets (A-1~A-6; indicated by black boxes). Movie available as Movie 1 (time in minutes post-inoculation). (B) Light microscope images showing hemocytes cultured with Sephadex beads. Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. Over time, the Sephadex beads (SP1, SP2, SP3, and SP4) became surrounded by hemocytes and encapsulated by various types of nets (B-1~B-9; indicated by black arrows). Movie available as Movie 2 (time in minutes postinoculation). Scale bar = 25 µm (A and B). Movie C shows hemocytes cultured alone, without Sephadex beads. Most hemocytes were actively moving. A few cells were attached to the culture slides by plasma membrane nets. However, large clusters of hemocytes were not observed.
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In this study, more than 1,000 cricket (Gryllus bimaculatus) hemocytes were classified based on their size and morphology. These hemocytes were classified into six types: granulocytes, plasmatocytes, prohemocytes, spherulocytes, coagulocytes, and oenocytoids. Hemocyte cultures was observed in real time to determine which hemocytes were associated w...
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... hemocytes rarely aggregated or formed large clusters. Figure 2A shows hemocytes cultured with E. coli (see also Supplementary Movie 1). Some hemocytes were observed to be more aggregated and moving. ...
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... traps) were produced by specific hemocytes, and various hemocytes were gathered together by these nets to form large clusters ( Fig. 2A-1~A-6; amoeba-like hairs or extracellular traps indicated by black arrows). As shown in Fig. 2A-1, three groups of hemocytes (indicated by black circles) were ultimately drawn into one cluster by the nets ( Fig. 2A-5 and A-6). However, it was not possible to determine whether the gathering of the hemocytes described above was triggered by E. coli. To address this question, the hemocytes were activated with Sephadex beads (120 μm diameter), which can be easily observed with a DIC microscope (Fig. 2B, Supplementary Movie 2). As shown in Fig. 2A, the ...
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... were ultimately drawn into one cluster by the nets ( Fig. 2A-5 and A-6). However, it was not possible to determine whether the gathering of the hemocytes described above was triggered by E. coli. To address this question, the hemocytes were activated with Sephadex beads (120 μm diameter), which can be easily observed with a DIC microscope (Fig. 2B, Supplementary Movie 2). As shown in Fig. 2A, the Sephadex beads were captured by the nets generated by specific hemocytes ( Fig. 2B; nets indicated by black arrows in the yellow boxes). The Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, ...
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... by the nets ( Fig. 2A-5 and A-6). However, it was not possible to determine whether the gathering of the hemocytes described above was triggered by E. coli. To address this question, the hemocytes were activated with Sephadex beads (120 μm diameter), which can be easily observed with a DIC microscope (Fig. 2B, Supplementary Movie 2). As shown in Fig. 2A, the Sephadex beads were captured by the nets generated by specific hemocytes ( Fig. 2B; nets indicated by black arrows in the yellow boxes). The Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, and SP3 were drawn together and into the area ...
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... gathering of the hemocytes described above was triggered by E. coli. To address this question, the hemocytes were activated with Sephadex beads (120 μm diameter), which can be easily observed with a DIC microscope (Fig. 2B, Supplementary Movie 2). As shown in Fig. 2A, the Sephadex beads were captured by the nets generated by specific hemocytes ( Fig. 2B; nets indicated by black arrows in the yellow boxes). The Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, and SP3 were drawn together and into the area indicated by the yellow box. In addition, SP1 eventually became completely surrounded by ...
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... observed with a DIC microscope (Fig. 2B, Supplementary Movie 2). As shown in Fig. 2A, the Sephadex beads were captured by the nets generated by specific hemocytes ( Fig. 2B; nets indicated by black arrows in the yellow boxes). The Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, and SP3 were drawn together and into the area indicated by the yellow box. In addition, SP1 eventually became completely surrounded by various hemocytes (Fig. 2B-9). The bead labeled SP4 also became incorporated into the cluster with SP1, SP2, and SP3 ( Fig. 2B-4~B-9; indicated by the yellow box). Over time, all the ...
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... indicated by black arrows in the yellow boxes). The Sephadex beads around the hemocytes were randomly labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, and SP3 were drawn together and into the area indicated by the yellow box. In addition, SP1 eventually became completely surrounded by various hemocytes (Fig. 2B-9). The bead labeled SP4 also became incorporated into the cluster with SP1, SP2, and SP3 ( Fig. 2B-4~B-9; indicated by the yellow box). Over time, all the Sephadex beads (SP1, SP2, SP3, and SP4) became surrounded by many hemocytes. By contrast, individual hemocytes were observed to be in contact with SP5, but it did not get drawn into ...
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... labeled SP1, SP2, SP3, SP4, and SP5. As can be seen by comparing Fig. 2B-2 and B-3, SP1, SP2, and SP3 were drawn together and into the area indicated by the yellow box. In addition, SP1 eventually became completely surrounded by various hemocytes (Fig. 2B-9). The bead labeled SP4 also became incorporated into the cluster with SP1, SP2, and SP3 ( Fig. 2B-4~B-9; indicated by the yellow box). Over time, all the Sephadex beads (SP1, SP2, SP3, and SP4) became surrounded by many hemocytes. By contrast, individual hemocytes were observed to be in contact with SP5, but it did not get drawn into the area indicated by the yellow box even though it was in a similar position as SP1. Therefore, ...
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... with carboxylate-modified polystyrene latex beads for 12 h (Supplementary Movie 3). The nets produced by the hemocytes (indicated by black arrows) actively moved around the latex beads (indicated by red arrows) ( Fig. 3A-1). Over time, the beads were engulfed by the nets and eventually could be seen within the cytoplasm of the hemocytes (Fig. 3A-2~A-4). However, not every carboxylate-modified polystyrene latex bead that encountered a net was phagocytosed. Thus, the movement of the activated hemocytes did not seem to correspond precisely with the movement of the ...
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... using a high-magnification confocal microscope ( Fig. 3B). At 4 h post-infection, the carboxylate-modified polystyrene latex beads could be observed within certain hemocytes ( Fig. 3B-1; beads indicated by red arrows, and specific hemocytes indicated by white circles). We next observed which specific cells engulfed the latex beads in more detail (Fig. 3B-2~B-6). Figure 3B-2 shows resting granulocytes, which were usually round or oval in shape, with many polymorphic dark granules within the cytoplasm. At 2 h after stimulation with carboxylate-modified polystyrene latex beads, the granulocytes began to show morphological changes, including fan-like or amoeba-like protrusions of the plasma ...
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... larger and wider over time (Fig. 3B-4 and B-5). At 48 h post-stimulation, many latex beads had accumulated in the cytoplasm of the granulocytes, but the nets could no longer be seen, and many granulocytes seemed to be inert ( Fig. 3B-6; beads indicated by red arrows). www.nature.com/scientificreports www.nature.com/scientificreports/ As shown in Fig. 2, the granulocytes appeared to attach not only to other granulocytes, but also to different types of hemocytes using these nets. We did not observe phagocytosis in any other cell type except for a small number of plasmatocytes (data not shown). In addition, we did not observe any immunological activity or morphological changes in any ...
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... As shown in Fig. 4A-1, a green fluorescent signal (phagocytosed E. coli particles) was www.nature.com/scientificreports www.nature.com/scientificreports/ observed in the granulocyte cytoplasm immediately after injection of the particles. At the same time, a red fluorescent signal, which indicates activated lysosome formation, was also observed ( Fig. 4A-2). At 4 h post-injection, highly polymorphic vacuoles could be seen in many granulocytes (Fig. 4A-4 and A-5). Merged images of the green fluorescent signal (phagocytosed E. coli particles) and the red fluorescent signal (activated lysosomes) are shown ( Fig. 4A-6). At 12 h post-injection, the green fluorescent signal began to dim, while ...
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... fluorescent signal at 48 h post-infection. To confirm this, the nuclei of the granulocytes were stained with 4′,6-diamidino-2-phenylindole (DAPI) at 48 h post-infection. As shown in Fig. 5B-1, granulocytes with two nuclei were frequently observed. Occasionally, granulocytes containing more than two nuclei, or deformed nuclei, were also observed ( Fig. 5B-2 and B-3; indicated by white ...
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... accurately observe cellular defense mechanisms such as nodulation, encapsulation, and phagocytosis of pathogens by cricket hemocytes. Our observations suggest that the cellular defense mechanisms of the cricket include the formation of sticky nets (amoeba-like hairs, pseudopods, and extracellular traps) by specific hemocytes (granulocytes) ( Fig. 2A and B). The formation of these nets has also been reported in mammalian neutrophils 19,[32][33][34] . Human neutrophils are a type of granulocyte. They are www.nature.com/scientificreports www.nature.com/scientificreports/ the most numerous type of immune cell and are one of the cell types known to phagocytose pathogens 35 . Recently, the ...
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... phagocytes most similar to the mammalian macrophage, crystal cells secrete components necessary for the melanization of www.nature.com/scientificreports www.nature.com/scientificreports/ invading organisms, and lamellocytes are rarely seen in healthy larvae and are involved in the encapsulation of invading pathogens [39][40][41] . As shown in Figs. 2 and 3, the encapsulation of pathogens in the cricket also seems to depend on net formation by granulocytes. Although only granulocytes were observed to form nets, encapsulation of pathogens might involve all six types of hemocytes working together. To further investigate the immunological function of the other types of hemocytes, we need to ...
Citations
... This study investigated the effects of short-term or long-term priming in connection with immune cell activation in Gryllus bimaculatus. In the past, we conducted cellular immunity studies with various insects (21)(22)(23)(24). In particular, we reported that cricket granulocytes perform very active cellular immune responses (phagocytosis, encapsulation, and nodulation) by forming pod-, fan-, and web-structures on their cell membrane when they come into contact with invading pathogens (24). ...
... In the past, we conducted cellular immunity studies with various insects (21)(22)(23)(24). In particular, we reported that cricket granulocytes perform very active cellular immune responses (phagocytosis, encapsulation, and nodulation) by forming pod-, fan-, and web-structures on their cell membrane when they come into contact with invading pathogens (24). Based on these researches, we induced immune priming using non-pathogenic substances and reinjected active pathogens to measure survival rate, immune responses of granulocytes, and lysosomes activation among immune priming challenged groups. ...
... For analysis using an optical microscope (Leica ® DM2500 and Leica ® DMI 3000B), 1,000 hemocytes per group were photographed and hemocytes image database for each group was constructed (LMD application software version 6.1, 2048x1536 pixels). We used this image data to classify hemocytes by size and shape (21)(22)(23)(24). To analyze lysosome activation in granulocytes, we stained the hemocytes collected from each group using the acid affinity dye LysoTracker Red (7.5nM, ...
This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the killed Bt-primed group, suggesting a correlation between the heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the killed Bt-primed group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the PBS buffer-primed group exhibited extracellular DNA trap cell death (ETosis), while in the killed Bt-primed group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, the heat-killed Bt-primed group, the heat-killed E. coli-primed group, and the PBS-primed group were re-injected with live Bt 2 and 9 days post priming. Two days later, only the PBS-primed group displayed low survival rates. After injecting live Bt 9 days later, the heat-killed E. coli-primed group surprisingly showed a similarly low survival rate, while the heat-killed Bt-primed group exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.
... Neutrophils carry out their physiological roles through mechanisms such as phagocytosis, degranulation, and the release of NETs (Muqaku et al., 2020). The process of NET formation has been identified as a novel form of neutrophil cell death known as NETosis (Cho and Cho, 2019;Chen et al., 2022). ...
Background: Chemokines and NETosis are significant contributors to the inflammatory response, yet there still needs to be a more comprehensive understanding regarding the specific molecular characteristics and interactions of NETosis and chemokines in the context of acute pancreatitis (AP) and severe AP (SAP).
Methods: To address this gap, the mRNA expression profile dataset GSE194331 was utilized for analysis, comprising 87 AP samples (77 non-SAP and 10 SAP) and 32 healthy control samples. Enrichment analyses were conducted for differentially expressed chemokine-related genes (DECRGs) and NETosis-related genes (DENRGs). Three machine-learning algorithms were used for the identification of signature genes, which were subsequently utilized in the development and validation of nomogram diagnostic models for the prediction of AP and SAP. Furthermore, single-gene Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed. Lastly, an interaction network for the identified signature genes was constructed.
Results: We identified 12 DECRGs and 7 DENRGs, and enrichment analyses indicated they were primarily enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, TNF signaling pathway, and T cell receptor signaling pathway. Moreover, these machine learning algorithms finally recognized three signature genes (S100A8, AIF1, and IL18). Utilizing the identified signature genes, we developed nomogram models with high predictive accuracy for AP and differentiation of SAP from non-SAP, as demonstrated by area under the curve (AUC) values of 0.968 (95% CI 0.937–0.990) and 0.862 (95% CI 0.742–0.955), respectively, in receiver operating characteristic (ROC) curve analysis. Subsequent single-gene GESA and GSVA indicated a significant positive correlation between these signature genes and the proteasome complex. At the same time, a negative association was observed with the Th1 and Th2 cell differentiation signaling pathways.
Conclusion: We have identified three genes (S100A8, AIF1, and IL18) related to chemokines and NETosis, and have developed accurate diagnostic models that might provide a novel method for diagnosing AP and differentiating between severe and non-severe cases.
... In the present study, larvae spent three days in the live pseudostem of Thodan exhibited severe hemocytopenia with sharp increase on certain hemocytes such as granulocytes and oenocytes. Granulocytes are cells which are actively involved in cellular immune response such as nodulation, encapsulation, phagocytosis of pathogen and removal of necrotic cells (Youngwoo and Sayoull, 2019). Numerical increase of granulocytes in the hemolymph of O. longicollis larvae in Thodan may be for the removal of dead cells formed as a result of antixenosis by the resistant host plant. ...
Banana pseudostem borer (BPB) Odoiporus longicollis (Olivier) (Coleoptera, Curculionidae) is a serious pest of Musa cultivars. Experimental maintenance of larvae in the live pseudostem of cultivar Thodan, a resistant AAB Musa cultivar has resulted antixenosis followed by death of larvae within a week. Antixenosis was characterized by significant decrease of total hemocytes and sharp changes on the proportionate distribution of different types of hemocytes in larvae. As the number of plasmatocytes, prohemocytes, splenocytes and adipohemocytes decreased the number of granulocytes and oenocytes increased. Antixenosis also caused accumulation of 20-hydroxyecdysone (20E) and significant inhibition on the activities of trypsin like serine protease (TlSP) and phenoloxidase (PO). Phytochemical analysis of Thodan resulted characterization of three larvicides such as Betulinic acid (BA), Stigmasterol-3-O-glucoside (SOG) and Sulfoquinovosyl diacyl glycerol (SQDG), and the content in the pseudostem ranged 0.0027 to 0.007 per cent. All the three larvicides were highly toxic to the larvae with LD50 of 0.38 ppm for SOG, 0.41 ppm for SQDG and 0.83 ppm for BA. Simultaneous action of three larvicides in the live pseudostem resulted resistance in Thodan against infestation by BPB. Susceptible Nendran showed negligibly low content of SOG (0.0011%) and SQDG (0.0013%) but no detectable quantity of BA. Intoxication by all the three larvicides caused significant changes on the proportionate distribution of hemocytes, accumulation of 20-hydroxy ecdysone (20E) and inhibition of TlSP and PO, the enzymes involved in larval metamorphosis and cuticle sclerotisation. This study demonstrated that resistance of Thodan against O. longicollis is due to adverse effect of these larvicides on endocrine system, cuticle development and cytotoxicity of hemocytes. As these larvicide molecules are stable compounds, there is scope for them to be used as substitutes in place of deleterious insecticides for the management BPB.
... However, continued and persistent use of these insecticides can result in development of resistance in insects against these toxicants. Since, haemocytes perform various vital physiological functions of the body and play a significant role in the cellular defense, the current study evaluated the effect of β-cyfluthrin on the insect's haemocyte count to understand their involvement in immunity of insect (Cho & Cho, 2019). ...
Dysdercus koenigii is a major global pest of cotton that causes severe economic loss. Among several control measures, pyrethroids are frequently used toxicants because of high efficacy at low dosages and relative safety. Since haemocytes are biomarkers of the physiological response and immunity of insect which determine the insecticide efficacy, the current study assessed the effect of a pyrethroid, β-cyfluthrin, on the total and differential haemocyte counts of D. koenigii. Haemolymph was collected from the fifth instars after the topical application of β-cyfluthrin (0.8, 1.6, 3.2, 6.4 and 12.8 mg/L) on the thoracic tergum. The haemolymph of control nymphs revealed 5270 haemocytes/mm3 which decreased instantly by 1.4-3.1-fold on β-cyfluthrin exposure; more reduction observed at lower dosages. Increase in exposure duration and β-cyfluthrin dosages fluctuated the count considerably, eventually raising them at lower dosages and diminishing at higher dosages. Among five kinds of haemocytes recorded in the haemolymph, the β-cyfluthrin exposure increased %prohaemocytes count; diminished %granulocytes and %plasmatocytes count while spherulocyte and oenocyte counts were inconsistent. The alterations in haemocyte counts indicate the immunity response trigger in D. koenigii due to β-cyfluthrin-induced stress. Further investigations may decipher the mechanisms involved and help to formulate the strategies for its management in fields.
... For example, immune haemocytes of insects perform various cell-mediated immune responses (such as phagocytosis) and may also simultaneously produce and discharge antimicrobial peptides (AMPs) as they react to and fi ght against pathogens (Ioannis et al., 2021). In other words, while the humoral immune response is mainly mediated by various substances that are produced by the fat body in insects, haemocytes membranes to rapidly form a vast net (netting) and encapsulate pathogens (Cho & Cho, 2019). Such rapid changes in the morphology of the cell membrane are reported for haemocytes in many species (Giglio et al., 2008). ...
... These three types of haemocytes are thus presumed to have highly signifi cant roles. For example, plasmatocytes and granulocytes, which are the main haemocytes involved in immunity in insects, perform phagocytosis in many insects and are also known to mediate encapsulation by interacting with other haemocytes (Cho & Cho, 2019;Eleftherianos et al., 2021). Thus, when studying cell-mediated immune response in insects, it is fi rst necessary to accurately identify and characterize the target insect's haemocytes. ...
... Therefore, haemocytes in different insects are identifi ed using morphological features, which requires years of research experience. In this regard, we have longstanding experience in the classifi cation of haemocytes in various insects (Kwon et al., 2014;Hwang et al., 2015;Lee et al., 2016;Cho & Cho, 2019). As such, we are able to classify the seven types of insect haemocytes in any target species with considerable accuracy. ...
In this study, haemocytes present in Papilio hyperbius Linnaeus were identified and characterized. Six different types of haemocyte were recorded in the haemocoel of this species of insect: prohaemocytes, plasmatocytes, granulocytes, spherulocytes, adipohaemocytes and oenocytoids. Of these the granulocytes were found to be responsible for cell-mediated immune responses such as phagocytosis. Granulocytes that were exposed to immunity inducers (carboxylate-modified polystyrene latex beads [CLBs] and Escherichia coli) had fan-like or pod-like structures on their cell membranes. The lysosomes in granulocytes were activated 2 h after injection with E. coli and after 12 h, all granulocytes exhibited highly activated lysosomes. After 24 and 48 h, the lysosome activity in granulocytes decreased. Transmission electron microscopy revealed that phagocytosis, which was mediated by granulocytes in the early hours of the E. coli infection, led to the formation of one phagosome for one E. coli within the cytosol. Moreover, as time passed, endosomes or lysosomes of different size developed. Subsequently, the phagosomes and lysosomes fused and E. coli were eliminated. After this series of immune responses, the nuclei of the granulocytes were indistinct and their cellular activity decreased. Hence, as old immune cells were replaced by new ones, active and healthy immune haemocytes were presumed to be maintained in the hemocoel.
... These hemocytes are described in the hemolymph of Rhipicephalus microplus as well, but only PRs, GRs and PLs are observed in fungus-infected R. microplus (Fiorotti et al., 2019). In addition, hemocyte types have been reported in other species as well, including ADs in Lymantria dispar (Butt and Shields, 1996), vermicytes (VEs) in Diatraea saccharalis (Falleiros et al., 2003) and COs in Gryllus bimaculatus (Cho and Cho, 2019), but they were not observed in the YPM larval hemolymph. ...
The yellow peach moth (YPM), Conogethes punctiferalis, is a destructive insect pest of
maize in eastern China and adapts to diverse environments, especially against pathogens. In insects, innate immunity comprising both humoral and cellular defense responses, is the primary defense against invading microbial pathogens. In this study, we identified five types of circulating hemocytes from the hemolymph of YPM larvae and analyzed their alterations and functions in immune responses to the infection of Beauveria bassiana, an entomopathogenic fungus infesting many lepidopteran species. The identified hemocytes included prohemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Significant decreases of total and differential hemocyte counts were recorded over time in larvae, after they were injected with B. bassiana conidia. Additionally, hemocyte-mediated phagocytosis and nodulation were initiated in the hemolymph of larvae from the B. bassiana conidia challenge. The introduction of DEAE-Sepharose Fast Flow beads stained with Congo red also induced a strong encapsulation response in the larval hemolymph. Our observations unraveled the occurrence of phagocytosis, nodulation and encapsulation in the hemocoel of YPM larvae to fight against the fungal infection, and offer the first insight into the YPM immune system.
... In some Drosophila species, for instance, inhibition of lamellocyte and crystal cell production impairs encapsulation and melanisation responses, respectively, in infected hosts (Binggeli et al., 2014;Trainor et al., 2021). The same has been demonstrated in some Lepidopterans when plasmatocyte and granulocyte proliferation and activity were suppressed (Pech and Strand, 1996;Chiu and Govind, 2002;Cho and Cho, 2019). Coupled with the above mechanisms, interfering with hemocyte spreading and adhesion (Rizki and Rizki, 1994;Cho and Cho, 2019) reduces the hosts' encapsulation ability, making them more susceptible to parasitisation. ...
... The same has been demonstrated in some Lepidopterans when plasmatocyte and granulocyte proliferation and activity were suppressed (Pech and Strand, 1996;Chiu and Govind, 2002;Cho and Cho, 2019). Coupled with the above mechanisms, interfering with hemocyte spreading and adhesion (Rizki and Rizki, 1994;Cho and Cho, 2019) reduces the hosts' encapsulation ability, making them more susceptible to parasitisation. ...
The oriental fruit fly, Bactrocera dorsalis (Hendel), and marula fruit fly, Ceratitis cosyra (Walker), are major fruit-infesting tephritids across sub-Saharan Africa. Biological control of these pests using parasitic wasps has been widely adopted but with varying levels of success. Most studies investigating host-parasitoid models have focused on functional and evolutionary aspects leaving a knowledge gap about the physiological mechanisms underpinning the efficacy of parasitoids as biocontrol agents of tephritids. To better understand these physiological mechanisms, we investigated changes in the cellular immune responses of C. cosyra and B. dorsalis when exposed to the parasitic wasps, Diachasmimorpha longicaudata (Ashmaed) and Psyttalia cosyrae (Wilkinson). We found that B. dorsalis was more resistant to parasitisation, had a higher hemocyte count, and encapsulated more parasitoid eggs compared to C. cosyra, achieving up to 100% encapsulation when exposed to P. cosyrae. Exposing B. dorsalis to either parasitoid species induced the formation of a rare cell type, the giant multinucleated hemocyte, which was not observed in C. cosyra. Furthermore, compared to P. cosyrae-parasitized larvae, those of both host species parasitized by D. longicaudata had lower encapsulation rates, hemocyte counts and spreading abilities and yielded a higher number of parasitoid progeny with the highest parasitoid emergence (72.13%) recorded in C. cosyra. These results demonstrate that cellular immune responses are central to host-parasitoid interaction in tephritid fruit flies and further suggest that D. longicaudata presents greater potential as a biocontrol agent of B. dorsalis and C. cosyra in horticultural cropping systems.
... Micro-associated molecular patterns (MAMPs) and pathogen-associated molecular patterns (PAMPs), such as peptidoglycan (PGN), lipopolysaccharide (LPS), β-glucans, lipoproteins, CpG dinucleotides and flagellin, are molecular markers recognized by the insect innate immune system. There are various cellular immune responses of insect blood cells (haemocytes) and humoral immune responses mediated by various effector molecules, including antimicrobial peptides (AMPs) and the phenol oxidase (PO) cascade is part of the insect immune system (Janeway et al., 2002;Hoffmann, 2003;Cho & Cho, 2019). Humoral immune responses involving Toll and immune deficiency (IMD) pathways are mainly activated by insect pattern recognition receptors (PRRs) (Wang et al., 2019). ...
We cloned and sequenced full-length peptidoglycan recognition protein (PGRP)-like cDNAs, named PS PGRP-SA(a)-like, PS PGRP-SA(b)-like, PS PGRP-SB1-like and PS PGRP-SC-like, from Protaetia brevitarsis seulensis. The amino acid sequences of PS PGRPs share 32.03-47.93% homology with those of PGRP family members in insects and mammals, including humans. We identified a conserved consensus sequence for amidase activity (His; H-Tyr; Y-His; H-Thr; T-Cys; C) and residues for binding peptidoglycan (PGN), one of the major bacterial cell wall components, including Asp (D) and Phe (F) for Lys-type PGN; and Gly(G), Trp (W) and Arg (R) for DAP-type PGN. The topological structures of PS PGRP-SA(a)-like, PS PGRP-SA(b)-like and PS PGRP-SC-like proteins are structurally similar to those of Drosophila melanogaster PGRP-SA, which has three α-helices and six β-strands. The β-strands are located in a central region and helix α1 on the back and peripheral α2 and α3 helices are on the front. The three α-helices and six β-strands are also present in PS PGRP-SB1-like, but the topological structure differs from that of typical PGRP. Significantly increased levels of PS PGRP-SA (a)-like and PS PGRP-SA (b)-like mRNA were recorded when Gram-positive bacteria or yeast cells were injected into larvae. PS PGRP-SB1-like mRNA levels were up-regulated by infection by all three pathogens; however, expression of PS PGRP-SC-like mRNA was increased 20- or 30-fold only shortly after injection with Gram-negative bacteria.
... On the other hand, Dipetalogaster maxima (Uhler, 1894) is the biggest triatomine and it is found in dry rocky parts of the Southern area of the California peninsula (Mexico). This species harbors epidemiological interest given its ability to inhabit peridomestic and intradomestic rural areas [3][4][5]. ...
Hemocytes, the cells present in the hemolymph of insects and other invertebrates, perform several physiological functions, including innate immunity. The current classification of hemocyte types is based mostly on morphological features; however, divergences have emerged among specialists in triatomines, the insect vectors of Chagas’ disease (Hemiptera: Reduviidae). Here, we have combined technical approaches in order to characterize the hemocytes from fifth instar nymphs of the triatomine Dipetalogaster maxima. Moreover, in this work we describe, for the first time, the ultrastructural features of D. maxima hemocytes. Using phase contrast microscopy of fresh preparations, five hemocyte populations were identified and further characterized by immunofluorescence, flow cytometry and transmission electron microscopy. The plasmatocytes and the granulocytes were the most abundant cell types, although prohemocytes, adipohemocytes and oenocytes were also found. This work sheds light on a controversial aspect of triatomine cell biology and physiology setting the basis for future in-depth studies directed to address hemocyte classification using non-microscopy-based markers.
... In Coleoptera also, there are at least five types of haemocytes, namely prohaemocytes, oenocytoids, plasmatocytes, granulocytes and spherulocytes [48,49] comparable to certain Cyclorrhapha [50][51][52]. In some basal insects, like the cricket Gryllus bimaculatus [53] and the migratory locust L. migratoria [54], six types were recognized, namely granulocytes, plasmatocytes, prohaemocytes, coagulocytes, oenocytoids and spherulocytes. However, others used a different terminology to describe the different morphological types of haemocytes in six triatomines (Hemipteran) [55]. ...
The host defense of insects includes a combination of cellular and humoral responses. The cellular arm of the insect innate immune system includes mechanisms which are directly mediated by hemocytes (e.g., phagocytosis, nodulation, and encapsulation). In addition, melanization accompanying coagulation, clot formation, and wound healing, nodulation, and encapsulation processes lead to the formation of cytotoxic redox‐cycling melanin precursors and reactive oxygen and nitrogen species. However, demarcation between cellular and humoral immune reactions as two distinct categories is not straightforward. This is because many humoral factors affect hemocyte functions and hemocytes themselves are an important source of many humoral molecules. There is also a considerable overlap between cellular and humoral immune functions that span from recognition of foreign intruders to clot formation. Here we review these immune reactions starting with the cellular mechanisms that limit hemolymph loss and participate in wound healing and clot formation and advancing to cellular functions that are critical in restricting pathogen movement and replication. This information is important because it highlights that insect cellular immunity is controlled by a multilayered system, different components of which are activated by different pathogens or during the different stages of the infection.
