Long-term in-vitro culture and subculture of the hemocytes of swimming crab Portunus trituberculatus

Crab cell line,especially continuous crab cell line,can provide us a useful tool for studies on the virology,immunology,and molecular biology of crabs.However,no continuous crab cell line has been available due to the lacking of suitable medium and the occurrence of mitosis-arrest.In this study,long-term in vitro culture conditions for both two-(2D)and three-dimensions(3D)were successfully developed for the circulating hemocytes of swimming crab Portunus trituberculatus,designated as PTH cells.In 2D culture,a novel crab basic medium in osmolarity of 990–1100 mOsm/kg was optimized for the first time,which is different from Leibovitz's L-15 medium in mainly the components of amino acids,containing double strengths of the contents of free amino acid mixture in the crab serum.Then an optimal crab growth medium was developed by supplementing 5%fetal bovine serum,50-g/L yeast extract powder,20-μg/L basic fibroblast growth factor and epidermal growth factor into the optimal crab basic medium,and found that it could support a long-term survival of PTH cells in a healthy monolayer up to 347 days and partially break through the mitosis-arrest of crab cells evidenced by the obvious increase of proliferating potential detected in the 10-d primarily cultured PTH cells.These 2D cultured PTH cells could be successfully sub-cultured for 11 times by physical flushing method and well cryopreserved in liquid nitrogen.In 3D culture,using the same crab growth medium,the PTH cell aggregates could be easily formed and healthily maintained on the surface of solidified Matrigel or in the ultra-low-attachment plate with a survival rate of 50%–60%on Day 103.This work largely improved the primary culture and subculture of crab cells and will facilitate the establishment of continuous crab cell line.

Modulation by Biogenic Amines for the Hemocyte Count and Prophenoloxidase Exocytosis via Receptors in Litopenaeus vannamei

Hemocyte counts and phenoloxidase(PO)activity were examined after hemolymph being incubated in dopamine(DA),noradrenaline(NE)and serotonin(5-HT).Results showed that all the three biogenic amines(BAs)had a significant impact on total hemocyte count(THC),differential hemocyte count(DHC),and intracelluar and extracelluar phenoloxidase(PO)activity.Among these Bas,DA had the strongest effect on the above parameters,whereas 5-HT had the least effect.Preincubation with D1 receptor antagonist SCH23390,D2 receptor antagonist Sulpiride and 1:1 admixture of the two could significantly inhibit the effect of DA on these parameters.SCH23390 showed a stronger inhibitory effect than Sulpiride,and the admixture exhibited the strongest effect.These results suggested that the change of hemocyte count and activation of prophenoloxidase(proPO)system in Litopenaeusvan-namei hemocyte can be regulated by BAs,and DA modulates the two parameters via its receptors.

Effects of Destruxin A on Hemocytes Morphology of Bombyx mori

Destruxin A （DA）, a kind of cyclo-hexadepsipeptide isolated from entomopathogenic fungus, Metarhizium anisopliae, is an inhibitor of insect＇s immunity. But its mechanism has not been clarified yet. In this study, the effects of DA on morphologic changes of in vivo and in vitro hemocytes of silkworm, Bombyx mori, were investigated by means of inverted phase contrast microscopy （IPCM）, fluorescence microscopy （FCM） and environmental scanning electron microscopy （ESEM）. The results indicated that DA was cytotoxic to granulohemocytes （GR） and plasmatocytes （PL）. The LC 50 values of DA against in vitro GR and PL of silkworm were 68.77 and 84.11 μg mL-1, respectively. However, the hemocytes in vivo were more susceptible to DA, although at the extremely low dose of 10 μL of 12.5 μg mL-1 for each insect （i.e., 0.036 μg g-1 body weight, or approximately 0.25 μg mL-1 hemolymph）, DA could induce obviously morphologic alterations of hemocytes in vivo. The results imply that there might be some factors in silkworm＇s hemolymph, which influence the interaction of DA and hemocytes.

Effects of mosquito hemocytes on normal and degenerated oocysts of Plasmodium yoelii and their role in oocyst melanization

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Early Detection of White Spot Syndrome Virus(WSSV)in Isolated Hemocytes of Litopenaeus vannamei

To date, White Spot Syndrome (WSS) produced by the White Spot Syndrome Virus (WSSV) causes one of the most severe diseases infecting penaeid shrimps worldwide. Although a vast amount of studies has elucidated pathogenesis in live infection models, there is still little information about the interaction of WSSV infections using in vitro models in the whiteleg shrimp Litopenaeus vannamei (L. vannamei) hemocytes. In this study, a WSSV infection kinetics was performed using total hemocytes isolated from healthy L. vannamei organisms and maintained in in vitro conditions using isotonic solution for shrimp (ISS). The infected experimental cells received ≈ 30,000 viral copies of WSSV. The viability of the hemocytes (control and infected group) was measured during the kinetics with trypan blue exclusion method and cells were maintained up to 6 hpi (post-infection) with non-significant differences of viability between both groups. WSSV replication was assessed using RT- PCR at the RNA expression level of the early viral gene Ie1 and transcripts were detected as early as 30 min pi. Hemocytes from WSSV group showed disrupted integrity, degranulation and irregular shape. This study provides evidence of the capability of WSSV to infect and replicates in L. vannamei hemocytes using in vitro assays in short times as 30 min.

ON THE ULIRASTRUCTURE AND CLASSIFICATION OF THE HEMOCYTES OF PENAEID SHRIMP, PENAEUS VANNAMEI (CRUSTACEA, DECAPODA)

The ultrastructure of hemocytes of Penaeus vannamei was examined by transmissionelectron mircroscope. Three types of hemocytes were identified: hyaline cell, small-granule cell andlarge-granule cell. Hyaline cells are the smallest of the hemocytes, lack electron-dense granules, haveaverage size of 11. 11 μp (SE ± 0.50, n = 10) × 6.00 μp (SE ± 0.55, n = 10), comprise 25. 8%(SE ± 2.87, n = 4 ×10) of the hemocyt; small granule cells are the mos abundan type of hamocytes,cornprise 58. 1% (SE ± 3.4o, n = 4 ×10) of the hees, contaln sInall electron - dense ,haVe average sbe of 10.78 mp (SE ± 0.00, n = 10) × 8.63 mp (SE ± 0.44, n = 10); and largn-gran-ule cells comprise 16. 1% (SE ± 2. 55, n = 4 × 10) of the hemocytes, are filled with large elecbondense guules, have average size of 12.51 μp (SE ± 0.63, n = 10) × 8.99μm (SE ±0.71, n = 10).

Morphofunctional characterization of hemocytes in black soldier fly larvae

In insects,the cell-mediated immune response involves an active role of hemo-cytes in phagocytosis,nodulation,and encapsulation.Although these processes have been well documented in multiple species belonging to different insect orders,information con-cerning 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 repre-sents 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 bac-terial 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,devel-oping 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 im-mune response by eicosanoids was investigated.

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx moil

Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace＇s medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs ofpndpS. Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.

Parasitism of Pieris rapae （Lepidoptera： Pieridae） by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae）： Effects of parasitism on differential hemocyte counts, micro- and ultra-structures of host hemocytes

Parasitism by the endoparasitic wasp Pteromalus puparum （Hymenoptera： Pteromalidae） by using only its associated venom, can suppress the immunal responses of Pieris rapae （Lepidoptera： Pieridae）. However, up to now, current knowledge of the mech- anisms has been limited. The response of host hemocytes to parasitism was investigated using a combination of light and transmission electron microscopy （TEM）. Five hemocyte types, prohemocytes （PRs）, granulocytes （GRs）, plasmatocytes （PLs）, oenocytoids （OEs） and coagulocytes （COs）, were observed and characterized from both unparasitized and parasitized Pieris rapae pupae. Light microscopy showed that both GRs and PLs became more round and spread abnormally after parasitism, whereas the shape of other types of hemocytes remained unaffected. In addition, the size of PRs and PLs became larger while OEs became smaller. The proportion of PRs significantly increased after parasitism and that of PLs decreased by 43.9%, but there was no significant increase of GRs and OEs. TEM showed that all types of hemocytes except COs were damaged to various degrees after parasitism, especially resulting in electron opaque cytoplasm and nucleus, fewer cell organelles of rough endoplasmic reticulum, mitochondria and vesicles. Our results indicate that parasitism by P. puparum affects differential hemocyte counts and structures of host hemocytes, particularly for GRs and PLs, which may be the main cause of the parasitoid suppressing host cellular immune responses.

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry(SCMC)combines features of traditional flow cytometry(i.e.,fluorescence-activated cell sorting)with mass spectrometry,making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms.In this study,we optimized SCMC to analyze hemocytes of the Drosophila innate immune system.We used metal-conjugated antibodies(against cell surface antigens H2,H3,H18,L1,L4,and P1,and intracellular antigens 3A5 and L2)and anti-IgM(against cell surface antigen L6)to detect the levels of antigens,while anti-GFP was used to detect crystal cells in the immune-induced samples.We investigated the antigen expression profile of single cells and hemocyte populations in naive states,in immune-induced states,in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase(hopTum)and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1[l(3)mbn1],as well as in stem cell maintenance-defective hdcD84 mutant larvae.Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes,plasmatocytes,and crystal cells,and delineated the unique immunophenotype of Drosophila mutants.We have identified subpopulations of L2^(+)/P1^(+)and L2^(+)/L4^(+)/P1^(+)transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum,respectively,and a subpopulation of L4^(+)/P1^(+)cells upon immune induction.Our results demonstrated for the first time that SCMC,combined with multidimensional bioinformatic analysis,represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

Development of two continuous hemocyte cell sublines in the Asian corn borer Ostrinia furnacalis and the identification of molecular markers for hemocytes

Granulocytes and plasmatocytes play important roles in clearing foreign objects in insects,but it is difficult to distinguish between them in immune reactions.Based on the hemocyte cell line SYSU-OfHe-C established at our lab,two cell sublines,SYSU-OfHe-C Granulocyte(Gr cells)and SYSU-OHe-C Plasmatocyte(PI cells),which possess the morphological characteristics of granulocytes and plasmatocytes,respectively,were established.Gr and PI cells showed different behaviors in immune reactions,such as spreading,phagocytosis and encapsulation.PI cells were easier to spread,but Gr cells tended to undergo aggregation,indicating that they may take different strategies to clear foreign objects.These results also suggested that granulocytes and plasmatocytes may express some different proteins.By comparing the gene expression in cells from the two sublines,1662 differentially expressed genes were identified,and 13 out of 30 transmembrane proteins highly ex pressed in PI cells(six)or Gr cells(seven)were further screened and confirmed by reverse-transcription polymerase chain reaction(PCR).Finally,three transmembrane genes specifically expressed in Pl cells and two transmembrane genes specifically expressed in Gr cells were screened out based on their expressions in immune reactions by quantitative PCR analysis.These genes may potentially be used as molecular markers to distinguish between granulocy tes and plasmatocytes in Ostrinia fiurnacalis,and further to clarify the functions of immune hemocytes in cellular immune reaction such as encapsulation and so on.

Characterization and mode of action analysis of black soldier fly(Hermetia illucens)larva-derived hemocytes

With the growing importance of the black soldier fly(Hermetia illucens)for both sustainable food production and waste management as well as for science,a great demand of understanding its immune system arises.Here,we present the first description of the circulating larval hemocytes with special emphasis on uptake of microorganisms and distinguishing hemocyte types.With histological,zymographic,and cytometric methods and with a set of hemocyte binding lectins and antibodies,the hemocytes of H.illucens are identified as plasmatocytes,crystal cells,and putative prohemocytes.Total hemocyte counts(THC)are determined,and methods for THC determination are compared.Approximately 1100 hemocytes per microliter hemolymph are present in naive animals,while hemocyte density decreases dramatically shortly after wounding,indicating a role of hemocytes in response to wounding(and immune response in general).The determination of the relative abundance of each hemocyte type(differential hemocyte count,DHC)revealed that plasmatocytes are highly abundant,whereas prohemocytes and crystal cells make up only a small percentage of the circulating cells.Plasmatocytes are not only the most abundant but also the professional phagocytes in H.illucens.They rapidly engulf and take up bacteria both in vivo and in vitro,indicating a very potent cellular defense against invading pathogens.Larger bioparticles such as yeasts are also removed from circulation by phagocytosis,but slower than bacteria.This is the first analysis of the potent cellular immune response in the black soldier fly,and a first toolbox that helps to identify hemocyte(types)is presented.

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.

Hemocytes transcriptome profile of the Chinese mitten crab(Eriocheir sinensis)

The Chinese mitten crab(Eriocheir sinensis)possesses an open circulatory system,in which hemolymph moves through interconnected sinuses or spaces surrounding organs called hemocoels.The hemocytes are classified into hyalinocytes,semigranulocytes,and granulocytes;and limited transcriptomics research is available.In this study,the transcriptome of the crab E.sinensis hemocytes was characterized.A total of 14,380,229 clean reads representing a total of 4.31 Gb nucleotides dataset were produced.A total of 67,047 contigs were obtained and 12.49%(8375)and 9.74%(6533)of the contigs were matched to data publicly available from the GenBank nr nucleotide and Uniprot databases,respectively.Among these contigs,4344 contigs belong to three categories of Gene Ontology,126 contigs to 21 subcategories of KEGG,and 4962 contigs to 25 categories of the COG database.A total of 508,379 and 345 transcripts of the E.sinensis showed>40%sequence similarity with transcripts expressed in the vertebrate blood cells from tilapia(Oreochromis niloticus),Chinese softshell turtle(Trionyx sinensis)and chicken(Gallus gallus)respectively.A total of 53,077 single nucleotide polymorphisms were identified and 9912 SSRs for microsatellite mining were found.The most frequent repeat motifs detected were dinucleotide repeats,accounting for 37.52%of the total repeats.Our data provides an important gene resource for the exploitation of molecular marker-assisted breeding,immune mechanisms and conservation of germplasm in the crab E.sinensis.

血细胞计数仪分析血细胞时抗凝剂及时间对结果的影响

目的：解读肝素、EDTA—K2抗凝剂及标本采集、放置时间对血细胞分析仪计数WBC、RBC、PLT的影响。方法：取新鲜血和经肝素、EDTA—K2抗凝的静脉务于立即、60、90、120min在f-820上对WBC、RBC、PLT进行行计数。结果：WBC、RBC用肝素抗凝剂在120min内与新鲜血没有显著性差异(P>0．05)，PLT用肝素抗凝剂在60min内与新鲜血无显著性差异(P>0．05)，60min后有显著性差异，用EDTA—K2 120min内无显著性差异(P>0．05)。

The insect cellular immune response

The innate immune system of insects is divided into humoral defenses that include the production of soluble effector molecules and cellular defenses like phagocytosis and encapsulation that are mediated by hemocytes. This review summarizes current understand- ing of the cellular immune response. Insects produce several terminally differentiated types of hemocytes that are distinguished by morphology, molecular and antigenic markers, and function. The differentiated hemocytes that circulate in larval or nymphal stage insects arise from two sources： progenitor cells produced during embryogenesis and mesodermally derived hematopoietic organs. Regulation of hematopoiesis and hemocyte differentiation also involves several different signaling pathways. Phagocytosis and encapsulation require that hemocytes first recognize a given target as foreign followed by activation of downstream signaling and effector responses. A number of humoral and cellular receptors have been identified that recognize different microbes and multicellular parasites. In turn, activation of these receptors stimulates a number of signaling pathways that regulate different hemocyte functions. Recent studies also identify hemocytes as important sources Of a number of humoral effector molecules required for killing different foreign invaders.

Insect immune resistance to parasitoids

Insect host-parasitoid interactions involve complex physiological, biochemical and genetic interactions. Against endoparasitoids, immune-competent hosts initiate a blood cell-mediated response that quickly destroys the intruders and envelops them in a multilayered melanotic capsule. During the past decade, considerable progress has been made in identifying some of the critical components of the host response, mainly because of the use of efficient molecular tools. This review examines some of the components of the innate immune response of Drosophila, an insect that has served as an exceptionally good experimental model for studying non-self recognition processes and immune cell signaling mechanisms. Topics considered in this review include hematopoiesis, proliferation and adhesion of hemocytes, melanogenesis and associated cytotoxic molecules, and the genetic aspects of the host-parasitoid interaction.

Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.

The long-term immunological effects of alloferon and its analogues in the mealworm Tenebrio mofitor

The subject of this article is a search for the long-term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro-apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe（p-NH2）^1 ]- and [Phe（p-OMe） ^1 ]-alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria-challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide-untreated bacteria-challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide-induced separation ofhemocyte fragments may resemble the formation ofplatelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half-life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N-terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.