Identification and characterization of the bombykal receptor in the hawkmoth Manduca sexta.

Manduca sexta females attract their mates with the release of a species-specific sex-pheromone blend, with bombykal (E,Z)-10,12-hexadecadienal and (E,E,Z)-10,12,14-hexadecatrienal being the two major components. Here, we searched for the hawkmoth bombykal receptor in heterologous expression systems. The putative pheromone receptor MsexOr1 coexpressed with MsexOrco in Xenopus oocytes elicited dose-dependent inward currents upon bombykal application (10-300 µmol l-1), and coexpressed in HEK293 and CHO cells caused bombykal-dependent increases in the intracellular free Ca2+ concentration. In addition, the bombykal receptor of Bombyx mori BmOr3 coexpressed with MsexOrco responded to bombykal (30-100 µmol l-1) with inward currents. In contrast, MsexOr4 coexpressed with MsexOrco responded neither to bombykal (30-100 µmol l-1) nor to the (E,E,Z)-10,12,14-hexadecatrienal mimic. Thus, MsexOr1, but not MsexOrco and probably not MsexOr4, is the bombykal-binding pheromone receptor in the hawkmoth. Finally, we obtained evidence that phospholipase C and protein kinase C activity are involved in the hawkmoth's bombykal-receptor-mediated Ca2+ signals in HEK293 and CHO cells.

Predator exposure-induced immunosuppression: trade-off, immune redistribution or immune reconfiguration?

Although predator exposure increases the risk of wound infections, it typically induces immunosuppression. A number of non-mutually exclusive hypotheses have been put forward to explain this immunosuppression, including: trade-offs between the immune system and other systems required for anti-predator behaviour, redistribution of immune resources towards mechanisms needed to defend against wound infections, and reconfiguration of the immune system to optimize defence under the physiological state of fight-or-flight readiness. We tested the ability of each hypothesis to explain the effects of chronic predator stress on the immune system of the caterpillar Manduca sexta Predator exposure induced defensive behaviours, reduced mass gain, increased development time and increased the concentration of the stress neurohormone octopamine. It had no significant effect on haemocyte number, melanization rate, phenoloxidase activity, lysozyme-like activity or nodule production. Predator stress reduced haemolymph glutathione concentrations. It also increased constitutive expression of the antimicrobial peptide attacin-1 but reduced attacin-1 expression in response to an immune challenge. These results best fit the immune reconfiguration hypothesis, although the other hypotheses are also consistent with some results. Interpreting stress-related changes in immune function may require an examination at the level of the whole organism.

Segmental pairs of giant insect cells discharge presumptive immune proteins at each larval molt.

A pair of massive secretory cells exists within each thoracic and the nine abdominal segments of Manduca larvae. Each of these cells is nestled between the dorsal integument and underlying muscles. Contents of large vacuoles in these cells are abruptly discharged at each molt and have always been considered to contribute to shedding and/or formation of cuticle. Peanut agglutinin is a specific lectin label for these secretory vacuoles; vacuoles label intensely immediately before each molt as vacuoles attain their maximal size. Contents of vacuoles are restored after each molt and throughout most of each intermolt. During the molt cycle these cells secrete contents of their vacuoles into the interior hemocoel rather than onto the exterior cuticle. Vacuoles discharge via a distinctive mechanism involving partitioning of contents into numerous vesicles that move to the cell surface. Dermal secretory cells were dissected from larvae before and after the 4th-5th instar molt. Proteins from pre-molt and post-molt secretory cells were separated by two-dimensional electrophoresis to establish which proteins are discharged at the molt. While secreted proteins are novel, all have presumptive roles in immune responses. Dermal secretory cells may represent a new, unsuspected component of the innate immune system that release their proteins during the vulnerable molting period of an insect's life.

Orientation-dependent changes in single motor neuron activity during adaptive soft-bodied locomotion.

Recent major advances in understanding the organizational principles underlying motor control have focused on a small number of animal species with stiff articulated skeletons. These model systems have the advantage of easily quantifiable mechanics, but the neural codes underlying different movements are difficult to characterize because they typically involve a large population of neurons controlling each muscle. As a result, studying how neural codes drive adaptive changes in behavior is extremely challenging. This problem is highly simplified in the tobacco hawkmoth Manduca sexta, which, in its larval stage (caterpillar), is predominantly soft-bodied. Since each M. sexta muscle is innervated by one, occasionally two, excitatory motor neurons, the electrical activity generated by each muscle can be mapped to individual motor neurons. In the present study, muscle activation patterns were converted into motor neuron frequency patterns by identifying single excitatory junction potentials within recorded electromyographic traces. This conversion was carried out with single motor neuron resolution thanks to the high signal selectivity of newly developed flexible microelectrode arrays, which were specifically designed to record from M. sexta muscles. It was discovered that the timing of motor neuron activity and gait kinematics depend on the orientation of the plane of motion during locomotion. We report that, during climbing, the motor neurons monitored in the present study shift their activity to correlate with movements in the animal's more anterior segments. This orientation-dependent shift in motor activity is in agreement with the expected shift in the propulsive forces required for climbing. Our results suggest that, contrary to what has been previously hypothesized, M.sexta uses central command timing for adaptive load compensation.

Physiological organization and topographic mapping of the antennal olfactory sensory neurons in female hawkmoths, Manduca sexta.

The hawkmoth, Manduca sexta, has been a keystone system for developmental, neurobiological, and ecological studies for several decades. Because many of its behaviors are driven by olfactory cues, a thorough understanding of the Manduca olfactory system is essential to studying its biology. With the aim of functionally characterizing single antennal olfactory sensory neurons (OSNs) and determining their detailed topographic location, we performed systematic single-sensillum recordings on 4 morphological types of olfactory sensilla: trichoid-A and -B and basiconic-A and -B. We were able to unambiguously differentiate the colocalized cells associated with single sensilla based on their spike amplitudes. Using a panel of 61 biologically relevant compounds established in behavioral and gas chromatography-electrophysiology experiments, we made 223 recordings from these sensilla. Based on the response spectra of 187 responding OSNs, the sensilla fell into 12 distinct functional classes encompassing 29 OSNs. Selectivity of the 25 responding OSNs varied from narrowly tuned (responding to only one or a subset of compounds), to very broadly tuned (responding to multiple compounds), in a concentration-dependent manner. Four OSNs, however, did not respond to the tested components. Topographic mapping of the sensilla revealed that some physiological sensillum types are confined to particular locations on the antennal surface while other classes are more or less irregularly scattered all over the antennal annuli. Such information will prove beneficial for future receptor deorphanization, in situ hybridization, and molecular manipulation experiments.

In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cß. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

Development of polyploidy of scale-building cells in the wings of Manduca sexta.

The developing wings of butterflies and moths are composed of two epithelial monolayers. Each epithelial sheet is made up of two kinds of cells, diploid cells that make up the epidermal surface and body of the wing, and large polyploid cells that become the scale-building cells whose cytoplasmic projections develop into the scales that will cover the adult wing and bear the pigment pattern. We studied the development of polyploidization of the scale-building cells during the pupal stage of the tobacco hornworm moth, Manduca sexta. The endomitotic divisions of the presumptive scale-building cells and the mitotic divisions of the diploid epithelial cells begin on day 3 of the pupal stage and continue until day 7. We show that scales of different colors and positions on the wing differ in size, and that the size of the scale is proportional to the ploidy of the scale-building cell. Scale-building cells are arranged in irregular rows and within each row there is an alternation of ploidy levels, with the lower ploidy cells giving rise to the underscales and the higher ploidy cells giving rise to the cover scales that carry the color pattern. Along the wing there is a proximo-distal decreasing gradient of average ploidy and scale size. Scale-building cells of high ploidy are surrounded by fewer epidermal cells than those of low ploidy. This inverse relationship is known as Henke's compensation principle, which posits that the number of endomitoses of a pre-polyploid cell and the number of mitotic divisions of its diploid daughter cell add up to a constant. We show that the inverse relationship fits the predictions of the compensation principle and does not fit constraints imposed by packing density, and we discuss mechanisms that could give rise to the inverse relationship.

A Toll-Spätzle pathway in the tobacco hornworm, Manduca sexta.

Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsToll(ecto) (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmToll(ecto) could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.

Wireless stimulation of antennal muscles in freely flying hawkmoths leads to flight path changes.

Insect antennae are sensory organs involved in a variety of behaviors, sensing many different stimulus modalities. As mechanosensors, they are crucial for flight control in the hawkmoth Manduca sexta. One of their roles is to mediate compensatory reflexes of the abdomen in response to rotations of the body in the pitch axis. Abdominal motions, in turn, are a component of the steering mechanism for flying insects. Using a radio controlled, programmable, miniature stimulator, we show that ultra-low-current electrical stimulation of antennal muscles in freely-flying hawkmoths leads to repeatable, transient changes in the animals' pitch angle, as well as less predictable changes in flight speed and flight altitude. We postulate that by deflecting the antennae we indirectly stimulate mechanoreceptors at the base, which drive compensatory reflexes leading to changes in pitch attitude.

Interaction of cellular and network mechanisms for efficient pheromone coding in moths.

Sensory systems, both in the living and in machines, have to be optimized with respect to their environmental conditions. The pheromone subsystem of the olfactory system of moths is a particularly well-defined example in which rapid variations of odor content in turbulent plumes require fast, concentration-invariant neural representations. It is not clear how cellular and network mechanisms in the moth antennal lobe contribute to coding efficiency. Using computational modeling, we show that intrinsic potassium currents (I(A) and I(SK)) in projection neurons may combine with extrinsic inhibition from local interneurons to implement a dual latency code for both pheromone identity and intensity. The mean latency reflects stimulus intensity, whereas latency differences carry concentration-invariant information about stimulus identity. In accordance with physiological results, the projection neurons exhibit a multiphasic response of inhibition-excitation-inhibition. Together with synaptic inhibition, intrinsic currents I(A) and I(SK) account for the first and second inhibitory phases and contribute to a rapid encoding of pheromone information. The first inhibition plays the role of a reset to limit variability in the time to first spike. The second inhibition prevents responses of excessive duration to allow tracking of intermittent stimuli.

Spiking patterns and their functional implications in the antennal lobe of the tobacco hornworm Manduca sexta.

Bursting as well as tonic firing patterns have been described in various sensory systems. In the olfactory system, spontaneous bursts have been observed in neurons distributed across several synaptic levels, from the periphery, to the olfactory bulb (OB) and to the olfactory cortex. Several in vitro studies indicate that spontaneous firing patterns may be viewed as "fingerprints" of different types of neurons that exhibit distinct functions in the OB. It is still not known, however, if and how neuronal burstiness is correlated with the coding of natural olfactory stimuli. We thus conducted an in vivo study to probe this question in the OB equivalent structure of insects, the antennal lobe (AL) of the tobacco hornworm Manduca sexta. We found that in the moth's AL, both projection (output) neurons (PNs) and local interneurons (LNs) are spontaneously active, but PNs tend to produce spike bursts while LNs fire more regularly. In addition, we found that the burstiness of PNs is correlated with the strength of their responses to odor stimulation--the more bursting the stronger their responses to odors. Moreover, the burstiness of PNs was also positively correlated with the spontaneous firing rate of these neurons, and pharmacological reduction of bursting resulted in a decrease of the neurons' responsiveness. These results suggest that neuronal burstiness reflects a physiological state of these neurons that is directly linked to their response characteristics.

Cell renewal in adjoining intestinal and tracheal epithelia of Manduca.

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects' responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.

Local interneuron diversity in the primary olfactory center of the moth Manduca sexta.

Local interneurons (LNs) play important roles in shaping and modulating the activity of output neurons in primary olfactory centers. Here, we studied the morphological characteristics, odor responses, and neurotransmitter content of LNs in the antennal lobe (AL, the insect primary olfactory center) of the moth Manduca sexta. We found that most LNs are broadly tuned, with all LNs responding to at least one odorant. 70% of the odorants evoked a response, and 22% of the neurons responded to all the odorants tested. Some LNs showed excitatory (35%) or inhibitory (33%) responses only, while 33% of the neurons showed both excitatory and inhibitory responses, depending on the odorant. LNs that only showed inhibitory responses were the most responsive, with 78% of the odorants evoking a response. Neurons were morphologically diverse, with most LNs innervating almost all glomeruli and others innervating restricted portions of the AL. 61 and 39% of LNs were identified as GABA-immunoreactive (GABA-ir) and non-GABA-ir, respectively. We found no correlations between odor responses and GABA-ir, neither between morphology and GABA-ir. These results show that, as observed in other insects, LNs are diverse, which likely determines the complexity of the inhibitory network that regulates AL output.

A subpopulation of mushroom body intrinsic neurons is generated by protocerebral neuroblasts in the tobacco hornworm moth, Manduca sexta (Sphingidae, Lepidoptera).

Subpopulations of Kenyon cells, the intrinsic neurons of the insect mushroom bodies, are typically sequentially generated by dedicated neuroblasts that begin proliferating during embryogenesis. When present, Class III Kenyon cells are thought to be the first born population of neurons by virtue of the location of their cell somata, farthest from the position of the mushroom body neuroblasts. In the adult tobacco hornworm moth Manduca sexta, the axons of Class III Kenyon cells form a separate Y tract and dorsal and ventral lobelet; surprisingly, these distinctive structures are absent from the larval Manduca mushroom bodies. BrdU labeling and immunohistochemical staining reveal that Class III Kenyon cells are in fact born in the mid-larval through adult stages. The peripheral position of their cell bodies is due to their genesis from two previously undescribed protocerebral neuroblasts distinct from the mushroom body neuroblasts that generate the other Kenyon cell types. These findings challenge the notion that all Kenyon cells are produced solely by the mushroom body neuroblasts, and may explain why Class III Kenyon cells are found sporadically across the insects, suggesting that when present, they may arise through de novo recruitment of neuroblasts outside of the mushroom bodies. In addition, lifelong neurogenesis by both the Class III neuroblasts and the mushroom body neuroblasts was observed, raising the possibility that adult neurogenesis may play a role in mushroom body function in Manduca.

The cellular and physiological mechanism of wing-body scaling in Manduca sexta.

In animals, appendages develop in proportion to overall body size; when individual size varies, appendages covary proportionally. In insects with complete metamorphosis, adult appendages develop from precursor tissues called imaginal disks that grow after somatic growth has ceased. It is unclear, however, how the growth of these appendages is matched to the already established body size. We studied the pattern of cell division in the tobacco hornworm Manduca sexta and found that both the rate of cell division and the duration of growth of the wing imaginal disks depend on the size of the body in which they develop. Moreover, we found that both of these processes are controlled by the level and duration of secretion of the steroid hormone ecdysone. Thus, proportional growth is under hormonal control and indirectly regulated by the central nervous system.

A switch in the control of growth of the wing imaginal disks of Manduca sexta.

BACKGROUND: Insulin and ecdysone are the key extrinsic regulators of growth for the wing imaginal disks of insects. In vitro tissue culture studies have shown that these two growth regulators act synergistically: either factor alone stimulates only limited growth, but together they stimulate disks to grow at a rate identical to that observed in situ. It is generally thought that insulin signaling links growth to nutrition, and that starvation stops growth because it inhibits insulin secretion. At the end of larval life feeding stops but the disks continue to grow, so at that time disk growth has become uncoupled from nutrition. We sought to determine at exactly what point in development this uncoupling occurs. METHODOLOGY: Growth and cell proliferation in the wing imaginal disks and hemolymph carbohydrate concentrations were measured at various stages in the last larval instar under experimental conditions of starvation, ligation, rescue, and hormone treatment. PRINCIPAL FINDINGS: Here we show that in the last larval instar of M. sexta, the uncoupling of nutrition and growth occurs as the larva passes the critical weight. Before this time, starvation causes a decline in hemolymph glucose and trehalose and a cessation of wing imaginal disks growth, which can be rescued by injections of trehalose. After the critical weight the trehalose response to starvation disappears, and the expression of insulin becomes decoupled from nutrition. After the critical weight the wing disks loose their sensitivity to repression by juvenile hormone, and factors from the abdomen, but not the brain, are required to drive continued growth. CONCLUSIONS: During the last larval instar imaginal disk growth becomes decoupled from somatic growth at the time that the endocrine events of metamorphosis are initiated. These regulatory changes ensure that disk growth continues uninterrupted when the nutritive and endocrine signals undergo the drastic changes associated with metamorphosis.

Developmental changes in dendritic shape and synapse location tune single-neuron computations to changing behavioral functions.

During nervous system development, different classes of neurons obtain different dendritic architectures, each of which receives a large number of input synapses. However, it is not clear whether synaptic inputs are targeted to specific regions within a dendritic tree and whether dendritic tree geometry and subdendritic synapse distributions might be optimized to support proper neuronal input-output computations. This study uses an insect model where structure and function of an individually identifiable neuron, motoneuron 5 (MN5), are changed while it develops from a slow larval crawling into a fast adult flight motoneuron during metamorphosis. This allows for relating postembryonic dendritic remodeling of an individual motoneuron to developmental changes in behavioral function. Dendritic architecture of MN5 is analyzed by three-dimensional geometric reconstructions and quantitative co-localization analysis to address the distribution of synaptic terminals. Postembryonic development of MN5 comprises distinct changes in dendritic shape and in the subdendritic distribution of GABAergic input synapses onto MN5. Subdendritic synapse targeting is not a consequence of neuropil structure but must rely on specific subdendritic recognition mechanisms. Passive multicompartment simulations indicate that postembryonic changes in dendritic architecture and in subdendritic input synapse distributions may tune the passive computational properties of MN5 toward stage-specific behavioral requirements.

All domains of Cry1A toxins insert into insect brush border membranes.

A critical step in understanding the mode of action of insecticidal crystal toxins from Bacillus thuringiensis is their partitioning into membranes and, in particular, the insertion of the toxin into insect brush border membranes. The Umbrella and Penknife models predict that only alpha-helix 5 of domain I along with adjacent helices alpha-4 or alpha-6 insert into the brush border membranes because of their hydrophobic nature. By employing fluorescent-labeled cysteine mutations, we observe that all three domains of the toxin insert into the insect membrane. Using proteinase K protection assays, steady state fluorescence quenching measurements, and blue shift analysis of acrylodan-labeled cysteine mutants, we show that regions beyond those proposed by the two models insert into the membrane. Based on our studies, the only extended region that does not partition into the membrane is that of alpha-helix 1. Bioassays and voltage clamping studies show that all mutations examined, except certain domain II mutations in loop 2 (e.g. F371C and G374C), which disrupt membrane partitioning, retain their ability to form ion channels and toxicity in Manduca sexta larvae. This study confirms our earlier hypothesis that insertion of crystal toxin does not occur as separate helices alone, but virtually the entire molecule inserts as one or more units of the whole molecule.

Pharmacological investigation of protein kinase C- and cGMP-dependent ion channels in cultured olfactory receptor neurons of the hawkmoth Manduca sexta.

In the hawkmoth Manduca sexta, pheromone stimuli of different strength and duration rise the intracellular Ca2+ concentration in olfactory receptor neurons (ORNs). While second-long pheromone stimuli activate protein kinase C (PKC), which apparently underlies processes of short-term adaptation, minute-long pheromone stimuli elevate cyclic guanosine monophosphate (cGMP) concentrations, which correlates with time courses of long-term adaptation. To identify ion channels involved in the sliding adjustment of olfactory sensitivity, inside-out patch clamp recordings on cultured ORNs of M. sexta were performed to characterize Ca2+-, PKC-, and cGMP-dependent ion channels. Stepping to positive holding potentials in high intracellular Ca2+ elicits different Ca2+-dependent ion channels, namely small-conductance channels (2-20 ps), medium-conductance channels (20-100 ps), and large-conductance channels (>100 ps). Ion channels of 40, 60, and 70 ps opened after PKC activation, whereas 10- and >100-ps channels were observed less frequently. Application of 8-bromo cyclic guanosine monophosphate opened 55- and 70-ps channels and increased the open probability of >100-ps channels, whereas even in the presence of phorbol ester 40-ps channels were inhibited. Thus, cGMP elevations activate a different set of ion channels as compared with PKC and suppress at least one PKC-dependent ion channel.

Nuclear translocation of immulectin-3 stimulates hemocyte proliferation.

Immulectin-3 (IML-3) is a C-type lectin from the tobacco hornworm Manduca sexta that contains a motif (NWGV) similar to the BH1 motif (NWGR) of the mammalian galectin-3. IML-3 is synthesized in fat body and secreted into hemolymph, but can be translocated into hemocytes. In this study, we showed that IML-3 was predominantly localized to the nucleus of hemocytes and some metaphase, anaphase and telophase hemocytes from M. sexta larvae injected with bacterial lipopolysaccharide (LPS). IML-3 was detected in the membrane and soluble extracts of hemocytes, suggesting that it may be translocated into hemocytes via receptor-mediated endocytosis. To investigate the role of IML-3 translocation to the nucleus, we expressed recombinant wild-type IML-3 and a deletion mutant DeltaIML-3 that has the NWGV motif deleted in Drosophila S2 cells. We found that recombinant wild-type IML-3, but not DeltaIML-3, was localized to the nucleus of some S2 cells and also detected in the nuclear extract. Expression of recombinant wild-type IML-3, but not DeltaIML-3 or GFP, increased the number of proliferating S2 cells. Our results suggest that nuclear translocation of IML-3 may stimulate hemocyte proliferation.