Disruption of insect immunity using analogs of the pleiotropic insect peptide hormone Neb-colloostatin: a nanotech approach for pest control II

This work continues our studies on the pleiotropic activity of the insect peptide Neb-colloostatin in insects. In vivo immunological bioassays demonstrated that hemocytotoxic analogs of Neb-colloostatin injected into Tenebrio molitor significantly reduced the number of hemocytes in the hemolymph and impaired phagocytosis, nodulation and phenoloxidase activities in the insects. Among the analogs tested, [Ala1]-,[Val1]-, [Hyp4]- and [Ach4]-colloostatin were particularly potent in disrupting cellular immunity in larvae, pupae and adult insects. This result suggests that the most effective analogs showed increases in the bioactivity period in the hemolymph of insects when compared to Neb-colloostatin. Recently, we demonstrated that it is possible to introduce Neb-colloostatin through the cuticle of an insect into the hemolymph when the peptide is coupled with nanodiamonds. In this study, we showed that [Ala1]-, [Val1]-, [Hyp4]- and [Ach4]-colloostatin, when complexed with nanodiamonds, may also pass through the cuticle into the hemolymph and induce long-term impairments of immunity in T. molitor at all developmental stages. Studies on the tissue selectivity and effectiveness of Neb-colloostatin analogs and efficient methods for their introduction into insects may contribute to the development of eco-friendly pest control methods based on bioactive peptidomimetics.


Materials and methods
]-colloostatin) was performed using a standard Fmoc procedure on Wang resins according to a previously described method 40 .
The noncovalent interactions between ND surfaces and peptides were used for the physical functionalization of NDs for Neb-colloostatin, [ 4 ]-colloostatin were dissolved in a physiological saline of Tenebrio to yield stock solutions of 1 mg/ml. The prepared stock solutions were stored at -30 °C, and the working solutions were prepared in physiological saline just before use. The functionalized ND solutions were sonicated for 1 h before use to break up possible aggregates 39 . Insects. Tenebrio molitor used in the study were derived from a laboratory stock population maintained at the Department of Animal Physiology and Developmental Biology, Institute of Experimental Biology, Adam Mickiewicz University, Poznań, Poland. Insects were provided with ad libitum access to oat flakes and wheat flour with 5% w/v brewer's yeast and supplemented with fresh carrot twice a week. The control and experimental groups (larvae, pupae and adults) were kept at the same population density in separate plastic boxes in a climate chamber at a constant temperature of 26 ± 0.5 ºC, with a relative humidity of 60 ± 5% and a photoperiod of 12 h of light and 12 h of darkness. All beetles used in our experiments were derived from parents that were less than 1 month old to avoid development disorders in the offspring. For each assay, the immunological studies were carried out on fifteen 120-140-mg larvae, 3-day-old pupae and 4-day-old adult beetles, with three biological replicates for each of the treatments.
Immune response assays. After anesthesia with CO 2 , the T. molitor larvae, pupae and adults were split into  4 ]-colloostatin) were conjugated with ND and applied to the cuticle surfaces of larvae, pupae and adults to assess whether these conjugates impede phagocytosis, nodulation and phenoloxidase activity. After anesthesia, the control insects received 5 µl www.nature.com/scientificreports/ of saline, while the experimental insects were exposed to 5 µl of the ND-Neb-colloostatin, ND Testing the circulating hemocyte count. The number of hemocytes circulating in the hemolymph of larvae, pupae and adults of T. molitor was studied according to a previously described method 39 . After anesthesia, the saline-, Neb-colloostatin-and analog-treated insects were washed in 70% ethanol and rinsed in distilled water. To determine the CHCs, the collected hemolymph samples (2 µl) were diluted in 18 µl of physiological saline containing an anticoagulant buffer (4.5 mmol/l citric acid and 9 mmol/l sodium citrate, 5:1 v/v). Subsequently, the hemolymph samples were placed on a Bürker hemocytometer and analyzed under a light microscope. The CHC values were based on the number of hemocytes observed in 15 random squares in the diluted hemolymph samples and calculated by the following equation: where CHC-the circulating hemocyte count; a-the average number of hemocytes in one square; 10-the dilution of the hemolymph sample; 250-the coefficient used to calculate the number of hemocytes in 1 µl of the sample.
Phagocytosis. The phagocytic ability of the hemocytes isolated from the control and experimental groups of insects at various stages of development was studied in vivo with latex beads (Sigma-Aldrich L1030) according to previously described methods 39  Nodulation. The ability of hemocytes to form nodules in insects that have been experimentally infected with bacteria was studied according to a previously described method 41 . Two hours after the injection of Neb-colloostatin or an analog solution or after the topical application of an ND-peptide solution (ND-Neb-colloostatin, ND  (5 μl). Nodule formation was measured on the third day of the experiment. The beetles were dissected to expose the nodules on the dorsal side of the hemocoels. Each insect body was pinned to a SYLGARD R silicone-filled Petri dish, and the hemocoel was washed with physiological saline. Next, the fat body and alimentary canal were removed. Nodule formation was studied with an Olympus SZX 12 stereoscopic microscope (Olympus Co., Tokyo, Japan), and 3 images of each beetle were taken with an Olympus U-LH100HG digital camera (Olympus Co., Tokyo, Japan). The number of nodules on the dorsal side of the hemocoel was counted in all of the beetles.
Humoral immune response. PO activity in S. aureus-infected T. molitor larvae, pupae and adults injected with Neb-colloostatin or an analog solution or exposed to an ND-peptide solution (ND-Neb-colloostatin, ND

Results
CHC assay. The impacts of Neb-colloostatin and its analogs on the number of hemocytes in the hemolymph of T. molitor larvae, pupae and adults is shown in Fig. 1. In larvae and pupae, all tested peptides caused a significant decrease in the CHC value throughout the whole experiment (Fig. 1A    ]colloostatin-treated and subsequently bacterially challenged larvae, pupae and adult insects in comparison to the PO activity of the controls. We demonstrated significant changes in PO activity in the hemolymph of insects at all developmental stages following topical applications of all ND-peptide conjugates in comparison to the PO activity of the control larvae, pupae and adult insects (Fig. 4D-F). All the most potent analogs conjugated with NDs caused greater reductions in PO activity than ND-Neb-colloostatin did.

Discussion
In insects, the goal of the immune response is to quickly and efficiently remove pathogens or abiotic particles from the hemolymph 44 . Cellular immunity is tightly integrated with humoral immunity through the activity of the hemocytes circulating in the hemolymph of insects. The hemocytes phagocytose pathogens and other foreign bodies in response to infection and/or isolate the pathogens from other host cells through nodule formation or encapsulation. Phagocytosis is initiated by pattern-recognition receptors on the surface of hemocytes that recognize and bind to complementary molecules or their fragments that are present in an abiotic particle, pathogen www.nature.com/scientificreports/ or apoptotic body. During the immune recognition of pathogens by hemocytes, the hemocytes recognize complementary chemical signals from the pathogens, such as fungal-specific β-1,3 glucans, bacterial peptidoglycans and lipopolysaccharides, and the nucleic acids of bacteria and viruses 44 . The engulfment of a pathogen or abiotic particle by a phagocyte depends on the local polymerization of actin and the formation of F-actin microfilaments at the remodeling site of a fragment of the cell membrane, which then surrounds the absorbed object and forms a phagosome 45 . At the next stage of phagocytosis, pathogens are killed by reactive oxygen species (ROS), and the enzymatic degradation of pathogens takes place in phagosomes with the participation of enzymes such as V-ATPase, DNase and cathepsin 46 . It has been suggested that the hemocytes that effectively remove pathogens from the insect's hemolymph are plasmocytes 47 . However, our previous studies indicate that, in T. molitor, the ability to phagocytose latex beads is demonstrated not only by plasmatocytes but also by granulocytes and even prohemocytes (unpublished results). Regarding nodule formation, it involves a large number of hemocytes in the isolation and inactivation of the invading pathogens 44 . Granulocytes are responsible for the immobilization of pathogens in the hemolymph and the chemotaxis of plasmatocytes to the site of infection. Granulocytes release the content of their granules into the insect's hemolymph, and together with plasmatocytes, they form a multilayered nodule that isolates pathogens from other host cells 48 . During nodule formation, remodeling of the hemocytes in the cytoskeleton occurs because the hemocytes that were previously freely circulating in the insect's hemolymph must become adhesive cells at the site of infection. Hemocytes are also responsible for the synthesis and release of prophenyloxidase into the hemolymph of the insect, which, after activation in the hemolymph by serine proteases, is involved not only in the recognition, opsonization and killing of pathogens but also in the coagulation of the hemolymph, wound healing, and sealing of nodules and capsules that isolate pathogens from other cells of the insect 44,47 . The ability of insects to maintain an appropriate number of hemocytes in the hemolymph is therefore of key importance for the resistance of insects to pathogens and thus for their viability and survival.
As shown in Fig. 1, all tested analogs of Neb-colloostatin caused significant decreases in the number of hemocytes circulating in the hemolymph of T. molitor larvae, pupae and adults compared to the number of hemocytes observed in the control groups. The findings of this study showed that the analogs [Ala 1 ]-, [Val 1 ]-,   40 . It is known that under physiological conditions, pathogen-induced macrophages and neutrophils produce and release large amounts of reactive oxygen species (ROS) that destroy intruders 44,49 . Despite the essential role of mitochondrial ROS in the response of immune cells to invading pathogens, mitochondrial ROS may also permeabilize mitochondrial membranes when overproduced and may consequently induce apoptosis of cells or cause organ failure [50][51][52] . We showed that Neb-colloostatin, when applied at picomolar and nanomolar doses, induced degenerative changes in mitochondria caused by disruption of the mitochondrial membrane potential, activated caspases in the cytoplasm and caused changes in the chromatin and F-actin cytoskeleton of hemocytes 38 . The doses of Neb-colloostatin and its analogs used so far in our investigations presumably promote significant increases in ROS formation in mitochondria, which in turn may lead to the permeabilization of the mitochondrial membrane and, finally, to hemocyte apoptosis. We presume that the analogs studied in this work may produce even greater amounts of ROS in hemocytes than Neb-colloostatin does and therefore, have higher proapoptotic activities and are more effective in reducing the number of hemocytes circulating in the hemolymph of larvae, pupae and adult insects. It is also possible that the Neb-colloostatin doses used so far in our studies significantly exceed the physiological range of doses of this peptide in the hemolymph of the studied insect 36,38,39 .
If this hypothesis turns out to be true, it would be necessary to verify the opinion of Neb-colloostatin as an immunosuppressive peptide and consider the possibility that this peptide, when naturally produced at physiological doses in insect ovaries, may have an immunostimulatory effect on insects. However, further detailed research is needed to clarify this issue. We also showed in this study that following a significant decrease in the number of circulating hemocytes in the hemolymph of larvae, pupae, and adult insects injected with Neb-colloostatin analogs, the number of these cells in the hemolymph of insects at all developmental stages gradually increased in the time tested, but this increase did not compensate for the number of hemocytes previously lost by apoptosis. However, the exception to this rule are adult insects injected with [Ac-Ser 1 ]-, [Asp 1 ]-, [Glu 1 ]-colloostatin, in which the number of hemocytes in the hemolymph reached the control value on the third day of the experiment (Fig. 1). The rate of increase in a total hemocyte population in the hemolymph was dependent on the type of chemical modification of the Neb-colloostatin molecule and on the developmental stage of the insect (Fig. 1). We suggest that chemically modifying the Neb-colloostatin molecule by introducing Ac-Ser, Asp, Glu, Ala or Val at the 1st position of the peptide amino acid chain or Hyp, Ach or Ile in the 4th position may impede access of the proteases contained in the hemolymph to the peptide bonds in the molecules of these analogs, and thus, the half-life of these analogs in the hemolymph of the insect may be extended. The results of the CHC study show that among the tested analogs, [Ala1]-, [Val1]-, [Hyp4]-and [Ach4]-colloostatin were the most effective in lowering the number of hemocytes circulating in the hemolymph and the cellular and humoral immune responses in T. molitor, which may suggest that these compounds are the more stable in the hemolymph of the insect. The gradual increase in the number of hemocytes circulating in the hemolymph of larvae, pupae and adult insects observed in the course of the study may not only be the result of differences in the half-life of the peptides in the insect hemolymph but may also indicate a gradual release of tissue-settled hemocytes as well as new populations of these cells from the hematopoietic organs. Restoration of the number of hemocytes in insect hemolymph was also found in Bombyx mori larvae after Escherichia coli injections 53 . Recently, we showed a similar effect in T. molitor individuals injected with either a solution of another immunomodulatory insect peptide, alloferon, or one of its analogs. We demonstrated that one day after the injection of these peptides into adult insects, apoptosis of hemocytes was observed, but later, a new pool of healthy hemocytes appeared in the hemolymph of the insect 41 . These findings indicated that hemocytes could have been released from the hematopoietic organs of the insects and that tissue-settled hemocytes could also be released into the hemolymph 41 . In the current study, we also demonstrated that the rate of restoration of the pool of hemocytes circulating in the hemolymph depends on the developmental stage of the insect. The increase in the number of hemocytes in the hemolymph was slowest in pupae, faster in larvae and fastest in adult insects (Fig. 1). It was also showed in a study of the effectiveness of JH analogs to interfere with the normal course of insect metamorphosis that the developmental stage of T. molitor and the house fly, Musca domestica, most sensitive to these analogs was the pupa, and in the bug Oncopeltus fasciatus, the last stage of the nymph was observed to be most sensitive 54 . This is because, during metamorphosis, the structure of an insect's body changes dramatically at the pupal stage owing to the simultaneous processes of histolysis and histogenesis; therefore, regeneration of the pool of lost hemocytes in pupae is probably more difficult than in larvae and adult insects 48 . As shown in Figs. 2, 3, 4, the significant reductions observed in the number of hemocytes and the slowing of the hemocyte count recovery in the hemolymph at all stages of T. molitor development caused by the studied Nebcolloostatin analogs have serious consequences for the functioning of the immune systems of insects, expressed as a marked impairment of the cellular and humoral immune responses of the insects. We recently demonstrated that Neb-colloostatin significantly reduced latex bead phagocytosis, nodule formation and phenoloxidase activation in experimentally infected larvae, pupae and adults of T. molitor resulting from a pronounced decrease in the number of hemocytes in the hemolymph of the insects 39 . Therefore, it is not surprising that when larvae, pupae, and adult insects were treated with analogs that are more proapoptotic than Neb-colloostatin, reductions in the cellular and humoral immune responses of the insects were observed. Moreover, the study of the influence of Neb-colloostatin analogs on short-and long-term cellular immune responses in larvae, pupae and adult insects ]-colloostatin were the most effective analogs in reducing not only the number of hemocytes in the hemolymph but also latex bead phagocytosis and nodule formation at all stages of T. molitor development.
Due to the multidirectional activity of PO in insects, disruption of the activity of this enzyme not only impairs the killing of pathogens by inhibiting the synthesis of ROS and quinones but also reduces the synthesis of melanins that seal the structure of nodules 48,55,56 . The release of prophenoloxidase into an insect's hemolymph is mediated by eicosanoids, which induce the lysis of hemocytes synthesizing this proenzyme 57 . However, during hemocyte apoptosis, cell lysis does not take place, but the dying cells shrink and form apoptotic bodies; therefore, it can be assumed that hemocytes that have started apoptosis as a result of the action of all tested Neb-colloostatin analogs are unable to phagocytose latex beads, form nodules around bacteria, or synthesize or release prophenoloxidase into the hemolymph of T. molitor larvae, pupae and adults that were experimentally infected with bacteria. On the other hand, a long-term effect of the hemocytotoxic action of the tested analogs was a gradual increase in the number of hemocytes in the hemolymph of larvae, pupae and adults of T. molitor (Fig. 1) and presumably for this reason, the efficacy of these analogs in inhibiting the humoral immune response was lower and less varied (Fig. 4) than was their effectiveness in reducing the cellular immune response in the bacterially infected insects (Fig. 3). Taken together, the results of the immunological bioassays seem to support our earlier hypothesis 40 that the presence of hydrophilic Ser at position 1 of the Neb-colloostatin amino acid chain is not important for its biological activity. Additionally, this means that not only the electric charge but also the structure of the side chain of the N-terminal amino acid residue is important for the immunological activity of Neb-colloostatin.
In experimental entomology, the method of injecting peptide solutions directly into the hemolymph of an insect is widely used; this method avoids the proteolytic degradation of peptides in the insect's digestive tract. Thanks to this method, it is possible to study the relationship between the chemical structure and the physiological activity of a tested peptide. This scientific approach allows the identification of the organ specificity of a peptide and its derivatives as well as the identification of the core sequence of a peptide molecule responsible for the binding of the peptide to its receptor and thus responsible for its specific physiological activity in the tissues and organs of an insect 24,28,36,38,41 . In turn, understanding the peptide core sequence allows for appropriate chemical modifications of the peptide molecule to prevent rapid degradation of the obtained analogs and peptidomimetics by peptidases contained in the hemolymph, tissues and intestine of pest insects 19,23,28,35,37 . The use of peptide derivatives with increased activity relative to native peptides for pest control is an accepted idea and is used to a limited extent [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] . However, the future practical use of peptides or their protease-resistant analogs/ peptidomimetics to effectively reduce the viability, reproduction and development of insect pests requires the development of alternative methods for introducing these molecules into an insect's body that are as effective as injecting peptides into the hemolymph of an insect. In vitro studies on the passage of chemically modified insect peptides through the cuticle model isolated from Heliothis virescens, Periplaneta americana and Heliothis peltigera have already been undertaken and have demonstrated that amphiphilic analogs of insect pyrokinin neuropeptides and pheromone biosynthesis-activating neuropeptides showed enhanced abilities to transmigrate the cuticles of insects in physiologically significant quantities without losing their intrinsic biological activities 23,24 . Recently, we demonstrated that it is possible to introduce a native peptide through an insect cuticle using a nanotechnological approach. We effectively introduced NDs coupled with Neb-colloostatin through the cuticle into the hemolymph of T. molitor individuals, where the peptide exhibits specific activity in vivo; i.e., the peptide induces hemocyte apoptosis and consequently significantly reduces the number of hemocytes circulating in the hemolymph and inhibits the cellular and humoral immune responses in all developmental stages of the insects 39 . In the current study, we also introduced the most active Neb-colloostatin analogs in combination with ND transcutaneously into the hemolymph of T. molitor larvae, pupae and adults, where the peptides effectively interfered with the cellular and humoral immune responses of the insects. The fact that [Ala 1 ]-, [Val 1 ]-, [Hyp 4 ]-and [Ach 4 ]-colloostatin and their conjugates with NDs were the most active analogs compared with the parent compound suggests that the introduction of amino acid residues that reduce the flexibility of the peptide backbone by restricting conformational freedom may have a significant impact on the penetration of the cuticle of an insect and on the biological activities of Neb-colloostatin.

Conclusion
Taken together, our findings indicate that significant long-term decreases in the number of circulating hemocytes in the hemolymph of larvae, pupae and adult insects caused by strong hemocytotoxic analogs of Neb-colloostatin result in significant reductions in the cellular and humoral immune responses of T. molitor at all studied developmental stages. Among the tested Neb-colloostatin analogs, we identified four particularly active compounds, i.e., [Ala 1 ]-, [Val 1 ]-, [Hyp 4 ]-and [Ach 4 ]-colloostatin, which very effectively reduced the number of hemocytes circulating in the hemolymph and the short-and long-term cellular immune response in larvae, pupae and adult insects. The pronounced activity of these analogs is probably the result of not only their increased hemocytotoxicity but also the elevated stability of these compounds in the hemolymph of an insect. The modifications introduced to the Neb-colloostatin molecule, which significantly increased the activity of its analogs, did not adversely affect the transfer of these analogs in a complex with NDs through the insect cuticle into the hemolymph. This result confirms the possibility of the effective introduction of peptides through the cuticle and into the body of an insect while maintaining their specific activities. In this work, we did not investigate the effects of the tested analogs on insect oogenesis, embryogenesis and metamorphosis, but the pleiotropic activity of Nebcolloostatin suggests that its immunologically potent analogs may also more effectively inhibit ovarian development and oogenesis and may interfere with the embryogenesis and metamorphosis of insects. The identification www.nature.com/scientificreports/ of pleiotropic, hemolymph-stable and highly active analogs of Neb-colloostatin and effective methods for their introduction into insects could contribute to the development of environmentally friendly pest control methods.

Data availability
The datasets used during the current study are available from the corresponding author upon reasonable request.