Hemolymph Proteins and Functional Peptides

The embryonic development of all oviparous animals occurs isolated from the maternal body. The egg should therefore contain all necessary supplies for the development of the embryo until eclosion. This supply is called yolk and is composed of nucleic acids, proteins, lipids and carbohydrates, stored in an organized manner inside the egg. Most of the yolk protein components are taken up during oocyte development via receptor-mediated endocytosis. Major proteins accumulated by insect oocytes are the two hemolymph lipoproteins, vitellogenin (Vg) and lipophorin (Lp). Once sequestered, the Vg is sent to yolk bodies for processing and stored as vitellin (Vn), the main nutritional reserves for the future embryo, whereas Lp (the high density Lp, HDLp) after unloading lipids (which are stored as lipid droplets, the energy source for the embryo) is stored as a very HDLp (VHDLp). This chapter reviews the current state of knowledge on the major yolk protein precuros (YPPs), the Vg and Lp, in insects and their role in oogenesis. Besides the biochemical and molecular aspects, the hormonal regulation and the uptake mechanism of these YPPs by the developing oocytes through receptor-mediated endocytosis will be addressed. Also, we point out some of the important areas for future research.

Insect flight activity involves the mobilization, transport and utilization of endogenous energy reserves at extremely high rates. In insects engaging in long-distance flights, an elevated supply of fuel, particularly lipids, is required for extended periods of time to support the sustained activity of the flight muscles. These lipids are mobilized from triacylglycerol (TAG) stores accumulated in cytosolic lipid droplets of fat body cells. As a result of TAG mobilization, the concentration of diacylglycerol (DAG) in the insect blood (hemolymph) is increased progressively and gradually constitutes the principal fuel for flight. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine adipokinetic cells in the glandular lobes of the corpus cardiacum, play a crucial role in this process by controlling the process of lipolysis in the fat body and integrating flight energy metabolism. The onset of flight activity triggers the release of AKHs; the binding of these hormones to their G protein-coupled receptors at the fat body target cell membranes induces a number of coordinated signal transduction processes that ultimately result in the activation of fat body TAG lipolysis and the release of DAG on which long-distance flight is dependent. Recent data reveal that the mechanisms guiding mobilization of stored lipids, including the action of lipid droplet-associated TAG lipases, are conserved between insects and mammals. The transport of lipids in insect hemolymph requires specific lipoprotein carriers (lipophorins) that act as a lipid shuttle; the AKHinduced increase in DAG loading during flight activity results in reversible changes in the lipophorin particles, requiring the association of the exchangeable apolipoprotein, apolipophorin III (apoLp-III), to the particle surface to allow increased lipid uptake and efficient transport to the flight muscles.

Storage proteins with different physico-chemical characteristics have been identified in the hemolymph of Lepidoptera, most of them are hexamerins, namely, hexamers of subunits of ca. 80 kDa. According to phylogenetic analyses of aligned nucleotide sequences, hexamerins are classified into at least three sub-groups: arylphorins, which are rich in aromatic amino acids at over 15 mol %, highly methioninerich hexamerins with methionine at 5.8-12 mol % (H-MtH) and moderately methionine-rich hexamerins with methionine at 3.4-5.4 mole % (M-MtH). These are evolutionally related to arthropod hemocyanin, HMtH and M-MtH being judged to be derived from the same branch, which is diverged from the branch to arylphorin in the phylogenetic tree. The other type of storage protein is biliverdin-binding protein (BP), a dimer or tetramer of ca. 150 kDa subunits, with high density (1.26 g/ml); it belongs to the vitellogenin family, but studies on this type have been limited to only a few species.

In most species, two or three different types of storage proteins are present, but the stages of their syntheses differ by species. In general, storage proteins are most actively synthesized by the fat body during the feeding period in the last larval instar, are soon after secreted in the hemolymph, and then, during larvalpupal development, are partly or totally sequestered by the fat body, being stocked in the protein granules. Some of the methionine-rich hexamerins (MtHs) are synthesized specifically in females, or more actively in females than in males, and in these cases, fluctuating profiles of MtHs and/or tracer surveys support their being utilized for ovarian development after being hydrolyzed to amino acids.

New findings in the common cutworm, Spodoptera litura have been described, in which five storage proteins, that is, three hexamerins and two BPs, are sequestered by the fat body during larval-pupal development, but these proteins are immuno-histochemically detectable in other tissues like midgut, Malpighian tube, integument, dorsal vessels and pericardial cells. Tissue distribution profiles of these storage proteins greatly change during development in a manner specific to respective proteins; in the pupal stage, all of them become distributed in the imaginal buds, which differentiate to adult tissues, and three species of storage proteins are detectable in eggs. These results raise the possibility that some tissues other than fat body are involved in the synthesis of these proteins, which should function as amino acid reserves in a specific period of molting, metamorphosis or reproduction.

Innate immune systems, which are essential for all metazoans, provide the first-line of defense against pathogens and comprise mainly humoral and cellular immune responses. Over the last two decades, genetic studies from a model insect, the fruit fly Drosophila melanogaster, have significantly contributed to elucidate the complex molecular mechanisms underlying the activation of humoral innate immune responses from pathogen recognition by pattern recognition receptors (PRRs) such as peptidoglycan recognition proteins (PGRPs), infection-induced nuclear factor (NF)-kB intracellular activation, and to the subsequent induction of hundreds of effector genes, including antimicrobial peptides (AMPs). In addition, many biochemical studies in Drosophila as well as in other arthropods such as the horseshoe crab recently uncovered the detailed molecular mechanisms of hemocyte-mediated immune responses, such as hemolymph coagulation or melanization, which have led us to reconsider the importance of cellular immune responses. This review describes recent progress towards understanding the molecular mechanisms of these innate immune responses mainly from studies of Drosophila, horseshoe crab, or other arthropods, and discusses the fundamental similarities and differences from mammalian innate immune systems.

The results of recent investigations show that the systolic contractions of the insect heart, similarly to those of the human heart, are based on a purely myogenic principle. A number of compounds have been previously tested for cardiostimulating or inhibitory effects, using simple in vitro bioassays on insect hearts explanted into saline. Some three decades ago, when the regulation of the insect heart was expected to be neurogenic, the possibility of cardiostimulating action attracted the attention of workers searching for the possible biological roles of newly isolated and synthesized neuropeptides. The first reports on the positive cardiostimulating effect of insect neuropeptides involved proctolin and its synthetic derivatives. These peptides increased the rate of heartbeat of hearts explanted into saline in the American cockroach (Periplaneta americana) or in the mealworm (Tenebrio molitor). These in vitro effects were not specific to the peptides; they were also nonspecifically induced by a number of low molecular compounds (amino acids, monoamines). The reports on the cardiostimulating effects of proctolin were followed by similar reports of cardiostimulating activity of CCAP (Crustacean Cardioactive Peptide) and, especially, on the "most potent cardioactive peptide" corazonin, which was isolated from the brain of the cockroach. The "cardiostimulating" label became widely adopted to advertise the importance and prestige of what were otherwise marginally important, crude biochemical studies. A myth was created about the "potent cardiostimulating properties" of several structural types of neuropeptides prepared synthetically or isolated from the brain and corpora cardiaca of insects. More recently, the tentatively cardioactive properties of insect neuropeptides were carefully reinvestigated, using advanced electrocardiographic recording methods for the prolonged monitoring of heartbeat in the living body of Manduca sexta, P. americana, T. molitor and Drosophila melanogaster. It has been determined that the "cardioactive" insect neuropeptides, proctolin, CCAP, corazonin and presumably other neuropeptides as well, have no cardiostimulating property under physiological conditions.

The midgut is an important organ for insects not only because it occupies a large space in their hemocoel, or because it is a major part of the digestive tract; it also plays critical roles in other physiological regulation such as metabolism, immune response, homeostasis of electrolytes, osmotic pressure, circulation and more. Therefore disturbance of these functions could provide a target and strategy for future pest management practice. Entry of entomopathogenic microorganisms and penetration of pesticides are through this route, and Plasmodium penetrating through the peritrophic membrane of Anopheline mosquitoes is a crucial issue for malaria control. The midgut is a complex organ that undergoes a gross transformation at metamorphosis to support different feeding habits at different developmental stages. It consists of epithelium and basal lamina with endodermal and mesodermal origins, respectively and the latter is innervated, i.e., an ectodermal element. We have not yet reached complete elucidation and understanding of the mechanism of embryonic formation of the organ, the metamorphosis and the regulatory mechanisms for a variety of midgut house-hold physiological functions. Another complexity comes from the unusual extent of variabilities that occur in general morphology, egg size/yolk content, feeding habit, mode of metamorphosis, or kinds of stresses or symbiotic associations that may occur in various orders of insects. We do not know whether the Drosophila model that provides important knowledge concerning embryonic formation is applicable to other insects. This review focuses on functions of developmental genes, endocrine agents, and local secretagogues, that control regulatory mechanisms, particularly peptides secreted from epithelial paraneurons as well as growth factors and differentiation factors synthesized by non-paraneuronal midgut cells such as columnar cells, goblet cells, perhaps trachea, hemocytes or muscles or extramidgut tissues such as the fat body. Stress conditions and metamorphosis, epithelial-mesenchymal interactions and behavior of adhesion molecules should be included here but currently this aspect has received little attention.

The insect brain peptides that induce target tissues to secrete steroid hormones, the ecdysones, are quite different from each other in peptide sequences and structures. Therefore, it is probable that related differences in specific receptors for the peptides exist on target organs, allowing for specific patterns of ecdysteroid synthesis by those tissues coordinated with regulation of metamorphosis. We identified and characterized an ecdysiotropic peptide from brains of Lymantria dispar that induced sheaths of testes to secrete ecdysteroid in vitro. The ecdysteroid in turn promoted synthesis of at least one peptide by fat body and/or testis sheaths that promoted sperm maturation and male gonadal imaginal disc development in vitro. We have cultured midgut stem cells and identified two peptidic factors controlling stem cell multiplication: the fast-acting brain peptide Bombyxin and the slower-acting fat body peptide, alpha-arylphorin. It is not known how these factors enter midgut cells, how they are processed, or how they incite stem cells to divide. We have characterized 4 differentiation-promoting growth factors, the MDFs, from culture medium and hydrolyzed hemolymph. They direct differentiation to normal and abnormal midgut cell fates. Internal [Ca²⁺] may be a second messenger for MDFs. Antibodies to the MDFs have revealed MDFs 1-4 in columnar cells and MDF4 in secretory cells as well. MDF2 appeared in the gut lumen, and MDF 4 is released from midgut secretory cells to both gut lumen and hemolymph.

An insect’s ability to endure and recover from stress is dependent on the suite of behavioral, biochemical, molecular and physiological mechanisms at its disposal when conditions become unfavorable. Expression of proteins to lessen and repair stress-related damage represents one of the most common and critical response mechanisms used by insects to counter the damage caused by traumatic periods. This chapter provides a comprehensive review of the regulation and function of proteins, both conventional and unconventional (not typically associated with stress), that prevent, alleviate and repair damage caused by environmental stress in insects as well as closely-related arthropods. First, we discuss situations known to be traumatic for insects, particularly those that have been previously documented as leading to negative consequences such as mortality or reduced fecundity. Stress signaling pathways and transcriptional regulation of stress proteins in insects are discussed due to their importance for triggering the initiation and subsequent regulation of the insect stress response. Heat shock proteins are reviewed individually since their increase has been documented in many insect species during and after stress. A synopsis is provided of how antioxidant proteins act to prevent damage caused by reactive oxygen species. Cytoskeletal and membrane structure changes have been documented during stress, and functions of these alterations, particularly during dehydration and cold exposure, are reviewed. Fluid and metabolite movement within insects is extremely important during periods of cold and dehydration, and proteins involved in regulating these movements are discussed. Similarly, proteins that buffer damage when the hydration state of an insect is altered are described. Aging, metabolism and reproduction changes that have been tied to traumatic conditions and stress protein expression, particularly for Drosophila, are assessed. Lastly, we provide a brief overview of other factors (i.e. polyols and sugars) that function in conjunction with proteins to prevent damage during stress. The complexity of the insect stress response far exceeds the usual suspects and includes proteins normally thought to function during unstressful conditions or only in a housekeeping manner (unconventional stress proteins). Our aim is to provide a review of all proteins, not just those typically associated with traumatic conditions, involved during the tolerance to and recovery from stress within insects and their close relatives.

Insulin was discovered as a key regulator of blood sugar level in 1922. In invertebrates, an insulin-like peptide, now named as bombyxin, was first isolated from the silkworm, Bombyx mori, with its prothoracicotropic activity to the saturniid moth, Samia cynthia ricini, in 1984. After the isolation of the peptide, homologous peptides were found in multiple insect and crustacean species. The insulin signaling pathway has also been recognized as a widely conserved pathway among animals. The conservations of molecular structure and signal pathway indicate that insulin has been one of the most important hormones discovered thus far. In this review, we summarize the molecular aspects of insulin and insulin-like (-related) peptides and then focus on their physiological functions in respect of sugar and lipid metabolisms, hormone biosynthesis, and developmental control such as body size and lifespan. We will make it clear that insulin and insulin-like peptide of arthropods are not just the hormones that regulate blood sugar levels but the strategic key hormones of growth and development. The functions of insulin and insulin-like peptide have been evaluated for hypotrehalosemic and hypoglycemic activities, ecdysteroidogenesis, juvenile hormone synthesis and secretion, lipid metabolism. In addition, they have been now recognized as main regulators of growth, lifespan, and reproduction.

ENF peptides are a family of insect specific multifunctional peptides consisting of 23-25 amino acids. They are involved in the regulation of many important biological processes, such as immunity, development, homeostasis, hibernation, and therefore regarded as a group of insect cytokines. In this chapter, I briefly review the structure biological functions, and modes of action of the ENF peptides.