Sensory neurons that respond to sex and aggregation pheromones in the nymphal cockroach.

In the common pest cockroach, Periplaneta americana, behavioural responses to the sex and aggregation pheromones change in an age-dependent manner. Nymphs are attracted by the aggregation pheromone periplanolide-E (PLD-E) but not by the sex pheromone periplanone-B (PB) in faeces. Adults display prominent behaviours to PB but not to PLD-E. Despite the significant behavioural differences depending on postembryonic developmental stages, peripheral codings of the sex and aggregation pheromones have not been studied in the nymph of any insects as far as we know. In this study, we morphologically and electrophysiologically identified antennal sensilla that respond to PB and PLD-E in nymphal cockroaches. Although nymphs lacked the sex pheromone-responsive single-walled B (sw-B) sensilla identified in adult males, we found PB-responsive sensory neurons (PB-SNs) within newly identified sw-A2 sensilla, which exhibit different shapes but have the same olfactory pores as sw-B sensilla. Interestingly, PLD-E-responsive sensory neurons (PLD-E-SNs) were also identified in the same sensillar type, but PB and PLD-E were independently detected by different SNs. Both PB-SNs and PLD-E-SNs showed high sensitivity to their respective pheromones. The hemimetabolous insect nymph has an ability to detect these pheromones, suggesting that behaviours elicited by pheromones might be established in brain centres depending on postembryonic development.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Structure and postembryonic development of the intersegmental nodules in the non-muscular joints of the antennae in Rhodnius prolixus.

The antennae of Insecta consist of two basal segments and the distal annulated flagellum lacking intrinsic muscles. Non-muscular joints are important to preserve the flexibility and structure of the long heteropteran antennae which bear an intersegmental nodule on each non-muscular joint. Little is known about their properties or function. Here we characterize the structure and postembryonic development of the non-muscular joints of Rhodnius prolixus antennae. Using Scanning Electron Microscopy, we tracked the changes in shape and size of both intersegmental nodules during the course of the hemimetabolous insect life cycle. Using Atomic Force Microscopy, we established a qualitative correlation between the topography of the surface and the rigidity of the joint between pedicel and flagellum. Our results confirmed the presence of two sub-articulations on each non-muscular joint. Also, the two intersegmental nodules have different origins: the one between the two flagellar segments (intraflagelloid) is a sclerite already present from the early nymph, while the nodule between pedicel and flagellum (prebasiflagellite) originates by gradual separation of the proximal end of the basiflagellum during postembryonic development. Various changes occur in the non-muscular joints and segments of the antenna during the life cycle of R. prolixus.

Development of ovary structures in the last larval and adult stages of psyllids (Insecta, Hemiptera, Sternorrhyncha: Psylloidea).

The development and organization of the ovaries of ten species from four Psylloidea families (Psyllidae, Triozidae, Aphalaridae and Liviidae) have been investigated. The ovaries of the last larval stage (i.e. fifth instar) of all examined species are filled with numerous clusters of cystocytes which undergo synchronous incomplete mitotic division. Cystocytes of the given cluster are arranged into a rosette with polyfusome in the centre. These clusters are associated with single somatic cells. At the end of the fifth instar, the clusters begin to separate from each other, forming spherical ovarioles which are surrounded by a single layer of somatic cells. In the ovarioles of very young females all cystocytes enter the prophase of meiosis and differentiate shortly thereafter into oocytes and trophocytes (nurse cells). Meanwhile, somatic cells differentiate into cells of the inner epithelial sheath surrounding the trophocytes and into the prefollicular cells that encompass the oocytes. During this final differentiation, the trophocytes lose their cell membranes and become syncytial. Oocytes remain cellular and most of them (termed arrested oocytes) do not grow. In the ovarioles of older females, one oocyte encompassed by its follicle cells starts growing, still connected to the syncytial tropharium by a nutritive cord. After the short phase of previtellogenesis alone, the oocyte enters its vitellogenic the growth phase in the vitellarium. At that time, the second oocyte may enter the vitellarium and start its previtellogenic growth. In the light of the obtained results, the phylogeny of psyllids, as well as phylogenetic relationships between taxa of Hemiptera: Sternorrhyncha are discussed.

An Ixodes scapularis cell line with a predominantly neuron-like phenotype.

The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 ß-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.

Location, morphology and function of nephrocytes in termites.

Insect nephrocytes are cells bathed in hemolymph and considered to have an excretory function. These cells have ambiguous nomenclature and are understudied in termites. This study is the first report on the occurrence, morphology and function of nephrocytes in different termite castes. Cytological characteristics in specific developmental stages and castes enable physiological functions to be inferred. Perforate diaphragms indicate a role in filtration, while the extensive peripheral invaginations of the cell membrane suggest active endocytosis. A sequence of morphologies in putative digestive vacuoles infers a lysosomal system and the occurrence of phosphatases suggests a function involving detoxification of substances sequestered from hemolymph. Pericardical nephrocytes took up the dye trypan blue injected in live termites, suggesting their activity connected to the filtration of the hemolymph. Additionally, histochemical tests showed the existence of stored proteins in their cytoplasm. These cells present a well-developed Golgi apparatus and abundant rough endoplasmic reticulum, consistent with protein synthesis. This study highlights the importance of nephrocytes in Isoptera and opens perspectives for further research of these cells.

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus.

Differentiation and morphogenesis of Triatoma infestans (Klug 1834) female gonads. I- Post embryonic development

The post-embryonic development of the female gonads in Triatoma infestans (Hemiptera, Heteroptera), insects of importance in health affairs as harbors and vectors of different tripanosomatidea flagellates, is presented in a complete follow-up since insect hatches from the egg up to the last molt in the fifth instar stage. The detailed description of the morphological changes which occur in each instar as well as careful measurements evaluating its size increase have been analyzed by stereomicroscopy, phase contrast, dark field, and oblique ilumination, in order to optimize the observations as well the photographic register of gonad morphology and structure. The analysis was performed on gonad specimens obtained from broods no less than twenty (20) nymph bugs, reared at constant temperature and fed-up regularly. According to the results of our study we can assert that gonad differentiation takes place in early phases of the insect development. Such is tge case that first instars nymph´s present absolutely and easily recognizably male and female gonads. From the third instar on beside the filament region, the three zones in each ovariole body is distinguished, a differentiation that is more noticeable during the fourth instar where a definite organization is present at the vitelarium. Such a clear cut zone development continues intensively during the fifth instar. Finally at the end of such fifth nymph stage and when the last molt toward adults is prepared, clear signs of ovariole maturation take place since oocytes in early vitelogenesis are found.

Differentiation and morphogenesis of Triatoma infestans (Klug 1834) female gonads. I- Post embryonic development

The post-embryonic development of the female gonads in Triatoma infestans (Hemiptera, Heteroptera), insects of importance in health affairs as harbors and vectors of different tripanosomatidea flagellates, is presented in a complete follow-up since insect hatches from the egg up to the last molt in the fifth instar stage. The detailed description of the morphological changes which occur in each instar as well as careful measurements evaluating its size increase have been analyzed by stereomicroscopy, phase contrast, dark field, and oblique ilumination, in order to optimize the observations as well the photographic register of gonad morphology and structure. The analysis was performed on gonad specimens obtained from broods no less than twenty (20) nymph bugs, reared at constant temperature and fed-up regularly. According to the results of our study we can assert that gonad differentiation takes place in early phases of the insect development. Such is tge case that first instars nymph s present absolutely and easily recognizably male and female gonads. From the third instar on beside the filament region, the three zones in each ovariole body is distinguished, a differentiation that is more noticeable during the fourth instar where a definite organization is present at the vitelarium. Such a clear cut zone development continues intensively during the fifth instar. Finally at the end of such fifth nymph stage and when the last molt toward adults is prepared, clear signs of ovariole maturation take place since oocytes in early vitelogenesis are found. (AU)

Differentiation and morphogenesis of Triatoma infestans (Klug 1834) female gonads. I--post embryonic development.

The post-embryonic development of the female gonads in Triatoma infestans (Hemiptera, Heteroptera), insects of importance in health affairs as harbors and vectors of different tripanosomatidea flagellates, is presented in a complete follow-up since insect hatches from the egg up to the last molt in the fifth instar stage. The detailed description of the morphological changes which occur in each instar as well as careful measurements evaluating its size increase have been analyzed by stereomicroscopy, phase contrast, dark field, and oblique illumination, in order to optimize the observations as well the photographic register of gonad morphology and structure. The analysis was performed on gonad specimens obtained from broods no less than twenty (20) nymph bugs, reared at constant temperature and fed-up regularly. According to the results of our study we can assert that gonad differentiation takes place in early phases of the insect development. Such is the case that first instars nymph's present absolutely and easily recognizably male and female gonads. From the third instar on beside the filament region, the three zones in each ovariole body is distinguished, a differentiation that is more noticeable during the fourth instar where a definite organization is present at the vitelarium. Such a clear cut zone development continues intensively during the fifth instar. Finally at the end of such fifth nymph stage and when the last molt toward adults is prepared, clear signs of ovariole maturation take place since oocytes in early vitelogenesis are found.

Postembryonic changes in the response properties of wind-sensitive giant interneurons in cricket.

The intensity-response (I-R) relations for four wind-sensitive giant interneurons (GIs 8-1, 9-1, 9-2 and 9-3) in the fourth-, sixth- and last-instar nymphs of the cricket, Gryllus bimaculatus, were investigated using a unidirectional air current stimulus in order to explore the functional changes of GIs during postembryonic development. Contrary to our expectations, the response properties of GIs in nymphs were largely different from those in adults. The response magnitude of GI 8-1 in an intact cricket decreased during development, i.e. the GI in younger insects showed a larger response magnitude. Although the response magnitudes of GIs 9-1 and 9-2 were almost identical during the nymphal period, a significant decrease was observed after the imaginal ecdysis. During the nymphal period, the response magnitude of GI 9-3 increased according to the developmental stage. However, it decreased significantly after the imaginal ecdysis. We also investigated the response magnitudes of the GIs in nymphs after unilateral cercal ablation. From the results of ablation experiments, the changes in excitatory and/or inhibitory connections between filiform hairs and each GI during postembryonic development were revealed.

Development of laminar organization in the mushroom bodies of the cockroach: Kenyon cell proliferation, outgrowth, and maturation.

The mushroom bodies of the insect brain are lobed integration centers made up of tens of thousands of parallel-projecting axons of intrinsic (Kenyon) cells. Most of the axons in the medial and vertical lobes of adult cockroach mushroom bodies derive from class I Kenyon cells and are organized into regular, alternating pairs (doublets) of pale and dark laminae. Organization of Kenyon cell axons into the adult pattern of laminae occurs gradually over the course of nymphal development. Newly hatched nymphs possess tiny mushroom bodies with lobes containing a posterior lamina of ingrowing axons, followed by a single doublet, which is flanked anteriorly by a gamma layer composed of class II Kenyon cells. Golgi impregnations show that throughout nymphal development, regardless of the number of doublets present, the most posterior lamina serves as the "ingrowth lamina" for axons of newborn Kenyon cells. Axons of the ingrowth lamina are taurine- and synaptotagmin-immunonegative. They produce fine growth cone tipped filaments and long perpendicularly oriented collaterals along their length. The maturation of these Kenyon cells and the formation of a new lamina are marked by the loss of filaments and collaterals, as well as the onset of taurine and synaptotagmin expression. Class I Kenyon cells thus show plasticity in both morphology and transmitter expression during development. In a hemimetabolous insect such as the cockroach, juvenile stages are morphologically and behaviorally similar to the adult. The mushroom bodies of these insects must be functional from hatching onward, while thousands of new neurons are added to the existing structure. The observed developmental plasticity may serve as a mechanism by which extensive postembryonic development of the mushroom bodies can occur without disrupting function. This contrasts with the more evolutionarily derived holometabolous insects, such as the honey bee and the fruit fly, in which nervous system development is accomplished in a behaviorally simple larval stage and a quiescent pupal stage.

Taurine-, aspartate- and glutamate-like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body.

The lobes of the mushroom bodies of the cockroach Periplaneta americana consist of longitudinal modules called laminae. These comprise repeating arrangements of Kenyon cell axons, which like their dendrites and perikarya have an affinity to one of three antisera: to taurine, aspartate, or glutamate. Taurine-immunopositive laminae alternate with immunonegative ones. Aspartate-immunopositive Kenyon cell axons are distributed across the lobes. However, smaller leaf-like ensembles of axons that reveal particularly high affinities to anti-aspartate are embedded within taurine-positive laminae and occur in the immunonegative laminae between them. Together, these arrangements reveal a complex architecture of repeating subunits whose different levels of immunoreactivity correspond to broader immunoreactive layers identified by sera against the neuromodulator FMRFamide. Throughout development and in the adult, the most posterior lamina is glutamate immunopositive. Its axons arise from the most recently born Kenyon cells that in the adult retain their juvenile character, sending a dense system of collaterals to the front of the lobes. Glutamate-positive processes intersect aspartate- and taurine-immunopositive laminae and are disposed such that they might play important roles in synaptogenesis or synapse modification. Glutamate immunoreactivity is not seen in older, mature axons, indicating that Kenyon cells show plasticity of neurotransmitter phenotype during development. Aspartate may be a universal transmitter substance throughout the lobes. High levels of taurine immunoreactivity occur in broad laminae containing the high concentrations of synaptic vesicles.

Flight muscle differentiation in nymphs of a dragonfly Anax imperator.

Flight muscle fibers of Anax imperator nymphs, in different developmental stages are analyzed for several morphological features, such as the arragnement and numerical ratio of actin and myosin filaments, the pattern of the T system and sarcoplasmic reticulum, the number of microtubules and the fractional volume of mitochondria in each fiber. The T system is initially represented by longitudinal grooves on the cell surface, joined with vesicles of the sarcoplasmic reticulum; this pattern rapidly changes and the grooves start to break up into longitudinal segments. The thin to thick filament ratio is at first quite high (about 4-4.5:1) but rapidly falls to the final (3:1) when the myofibrils are well developed at the fiber periphery. Statistical analyses show that the measured values are significantly different in the various stages of development, also indicating a progressive reduction of the ratio variability. The reduction of thin to thick filament ratio and the variance decrease fit quite well with the hypothesis that the synthesis of actin and myosin depends on independently regulated messenger RNA molecules.

Studies on the proteins of Poecilocera picta. I-changes taking place in the haemolymph protein during moulting.

The haemolymph proteins of the 6th nymph of P. picta were fractioned by the polyacrylamide gel disc electrophoresis. A total of seven proteins fractions have been detected from the haemolymph. The chemical nature of different protein fractions have been examined by histochemical methods. The changes taking place in the cuticle and epidermal cells have been examined during the transformation of 6th nymph into adult. The fat body proteins have been electrophoretically fractioned and the changes in the concentration of different protein fractions have been examined. It is suggested that the protein fraction 3 of the haemolymph is utilized in the formation of new cuticle. It is concluded by the histochemical observations that proteins of the band 3 are synthesized in the fat body.