Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

Digestive enzymes in larvae of the leaf cutting ant, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini)

The digestive physiology and biochemistry of larvae of the leaf-cutting ant Acromyrmex subterraneus were investigated here. The activity of digestive enzymes was evaluated in the labial glands, midgut epithelium (soluble and particulate fractions), and in the lumen contents, separated into endo and ectoperitrophic regions. Enzymes with high levels of activity were partially characterised using chromatography and electrophoresis techniques. Microscope observations were carried out and the anatomy of the larval digestive tract was described here for the first time. Larvae fed with pH indicator solutions showed the anterior portion of the midgut to be acidic and the posterior portion neutral to alkaline, indicating that the pH of the different regions of the midgut could optimise certain enzyme activities, whilst inhibiting others. The flow rate of the intestinal contents was also evaluated in larvae fed with a dye solution. The slow flow rate is probably due to closure of the rear end of the larval midgut. No compartmentalisation of digestive enzymes acting on oligosaccharides and disaccharides in the ectoperitrophic space and on polysaccharides in the endoperitrophic space was observed here, which could also be related to the closure of the midgut. The digestive physiology of these larvae is therefore similar to ancestral Holometabola, a paradox when considering the highly evolved nature of these insects. The larval midgut demonstrated a large diversity of enzyme activities with high levels of alpha-amylase, alpha-mannosidase, chitinase, alpha-glucosidase, beta-glucosidase and proteinase. High levels of chitinase and amylase activities were detected in the labial glands of larvae. The enzyme profile reflected the necessity of the larvae to degrade the fungal substrate, their sole diet, and a probable source of some of the digestive enzymes detected here. When compared to adults, the larvae had a greater diversity and higher levels of enzyme activity, highlighting their importance as the "digestive caste" of the colony.     

Digestive enzymes of leaf-cutting ants, Acromyrmex subterraneus (Hymenoptera: Formicidae: Attini): distribution in the gut of adult workers and partial characterization

Enzyme activities associated with the labial glands, midgut and rectum of adult Acromyrmex subterraneus were investigated in order to understand their role in digestion of plant and fungal material. High chitinolytic activity was detected in the labial glands, indicating a possible role in the degradation of fungus ingested by the ants. Chitinolytic activity seen in other compartments of the alimentary canal probably originated in the labial glands. The highest activity detected in the midgut was for alpha-glucosidase, which was considered to be of insect origin due to its association with midgut epithelium and it is probably involved in glucose assimilation from nutrient sources such as maltose and sucrose present in plant material. A large range of enzyme activities were detected in the rectal lumen contents, and as in the midgut the highest values were for alpha-glucosidase activity. The absence of activity associated with the epithelium, in the particulate fraction, indicates that the rectal epithelium does not have a secretory function. The detection of enzymes in the rectal lumen contents, which were not detected in the midgut lumen contents, indicates that the rectum acts as a reservoir, accumulating enzymes. The major digestive enzymes were partially characterized using hydrophobic interaction chromatography, gel filtration and SDS-PAGE. The pH of the adult intestinal tract and flow rate of dye through the tract was investigated. A gradual acidification of the intestinal tract was noted commencing with the crop (pH 6-8.2) and terminating with the rectum (pH 3-5). The flow of dye through the different compartments of the tract showed a rapid fill time for all the gut compartments and a short residence time in the crop. In all other compartments, the dye remained detectable for 10 days or longer.     

The larval midgut of the cassava hornworm (Erinnyis ello)

Summary Columnar cells of the larval midgut of the cassava hornworm, Erinnyis ello, display microvilli with vesicles pinching off from their tips (anterior and middle midgut) or with a large number of double membrane spheres budding along their length (posterior midgut). Basal infoldings in columnar cells occur in a parallel array with many openings to the underlying space (posterior midgut) or are less organized with few openings (anterior and middle midgut). Goblet cells have a cavity, which is formed by invagination of the apical membrane and which occupies most of the cell (anterior and middle midgut) or only its upper part (posterior midgut). The infolded apical membrane shows modified microvilli, which sometimes (posterior midgut) or always (anterior and middle midgut) contain mitochondria. The cytoplasmic side of the membrane of the microvilli that contain mitochondria are studded with small particles. The results suggest that the anterior and middle region of the midgut absorbs water, whereas the posterior region secretes it. This results in a countercurrent flux of fluid, which is responsible for the enzyme recovery from undigested food before it is expelled. Intermediary and final digestion of food probably occur in the columnar cells under the action of plasma membrane-bound and glycocalix-associated enzymes.     

Reproductive Isolation in the Wood Cricket Gryllus vernalis (Orthoptera: Gryllidae)

Two species of closely related wood crickets, Gryllus vernalis (Orthoptera: Gryllidae) and G. fultoni, occur together in the eastern USA and have a similar calling‐song structure, consisting of three‐pulse chirps. Previous studies revealed that male calling song and female selectivity were divergent between sympatric and far allopatric populations of G. fultoni, consistent with the pattern expected of reproductive character displacement. We studied the reproductive isolation of G. vernalis in relation to G. fultoni by investigating the geographic variation in calling songs of G. vernalis and by examining close‐range mating behaviors. Neither field nor laboratory studies revealed differences in any of the calling‐song characters between sympatric and allopatric populations, but this could reflect the limited sampling within the relatively small region of allopatry for G. vernalis. Although close‐range mating trials revealed that females of both species discriminated against heterospecific mating partners, the strength of discrimination was especially strong in females of sympatric G. vernalis populations. Our studies of long‐range and close‐range mating behaviors suggest that selection pressures for reproductive isolation are exerted primarily on close‐range mating behaviors in G. vernalis but on long‐range mating behaviors in G. fultoni.     

Characterization of arylalkylamine N-acetyltransferase (AANAT) activities and action spectrum for suppression in the band-legged cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae)

Arylalkylamine N-acetyltransferase (AANAT), constituting a large family of enzymes, catalyzes the transacetylation from acetyl-CoA to monoamine substrates, although homology among species is not very high. AANAT in vertebrates is photosensitive and mediates circadian regulation. Here, we analyzed AANAT of the cricket, Dianemobius nigrofasciatus. The central nervous system contained AANAT activity. The optimum pHs were 6.0 (a minor peak) and 10.5 (a major peak) with crude enzyme solution. We analyzed the kinetics at pH 10.5 using the sample containing collective AANAT activities, which we term AANAT. Lineweaver-Burk plot and secondary plot yielded a K_m for tryptamine as substrate of 0.42 µM, and a V_max of 9.39 nmol/mg protein/min. The apparent K_m for acetyl-CoA was 59.9 µM and the V_max was 8.14 nmol/mg protein/min. AANAT of D. nigrofasciatus was light-sensitive. The activity was higher at night-time than at day-time as in vertebrates. To investigate most effective wavelengths on AANAT activity, a series of monochromatic lights was applied (350, 400, 450, 500, 550, 600 and 650 nm). AANAT showed the highest sensitivity to around 450 nm and 550 nm. 450 nm light was more effective than 550 nm light. Therefore, the most effective light affecting AANAT activity is blue light, which corresponds to the absorption spectrum of blue wave (BW)-opsin.     

Wolbachia infection in the Loxoblemmus complex (Orthoptera: Gryllidae) in Korea

The Wolbachia bacterium is one of the most prevalent intracellular symbionts of invertebrates, particularly insects. This bacterium induces four distinct reproductive anomalies such as cytoplasmic incompatibility, feminization, male killing, and parthenogenesis of its hosts. Here we report that three closely related cricket species, Loxoblemmus doenitzi, L. campestris, and L. equestris can become infected with Wolbachia. Based on the 16s rRNA sequences, all three species were single infections. However, Wolbachia infecting L. campestris showed diverse Wolbachia surface protein gene sequences resembling multiple infections. In addition, all Wolbachia strains in the three host species harbored the Wolbachia specific bacteriophage.     

Mating in the Scaly Cricket Cycloptiloides canariensis (Orthoptera: Gryllidae: Mogoplistinae)

The process of mating in C. canariensis follows basically the same pattern as in other crickets: adoption of the female-above-male position, hooking of the male onto the female's subgenital plate, spermatophore transfer, and separation of the mates. Two crucial modifications can, however, be distinguished: Hooking is not accomplished by means of a sclerotized process from the protruded epiphallus, but with paired hooklets on the paraproct (paraproct processes; sternite 11). The paraproct processes are about 0.4 mm long and covered with bristles, and a group of campaniform sensilla is found in the tip region. The time course of copulation is also modified. Usually, in crickets an already fully formed spermatophore is transferred immediately after mounting, and remains attached for a considerable period. C. canariensis, however, needs about 15 min for spermatophore production, while the couple is already hooked. After transfer the spermatophore remains attached only for an average of 31 s. With both hooklets severed, copulation is unsuccessful. Severance of only one hooklet prolongs the initial hooking phase but shortens the following interval, which suggests that spermatophore production is triggered during a definite interval before hooking, and continues as an autonomous internal process.     

Characterization of an iridescent virus isolated from Gryllus bimaculatus (Orthoptera: Gryllidae)

We have isolated an iridescent virus from commercially produced colonies of Gryllus bimaculatus in Germany, which showed apparent mortality. Transmission electron microscopy studies on adult cricket specimens revealed the paracrystalline assembly of icosahedral virus particles in the cytoplasm of hypertrophied abdominal fat body cells. The infecting agent could be cultivated in the lepidopteran cell line sf-9, where it caused cytopathogenic effects such as cell hypertrophy, cytoplasmic vacuolization, and cell death within 8 days postinfection. Infection titers of the first virus passage reached 10(7.5) TCID(50)/ml. Negatively stained virus particles (n = 100) had dimensions of 172 +/- 6 nm (apex to apex) and 148 +/- 5 nm (side to side). SDS-polyacrylamide gel electrophoresis of virus proteins showed more than 20 distinct polypeptides with a major species of approximately 50 kDa. Analysis of the restriction fragment length profiles from digestion of purified viral DNA with the endonucleases EcoRI, BamHI, and HindIII showed marked differences from the profiles of iridoviruses of lower vertebrates (genus Ranavirus), e.g., Rana esculenta Iridovirus and Frog virus 3. Restriction enzyme digests with the endonucleases MspI and HpaII indicated the lack of methylation of viral DNA. Polymerase chain reaction led to the amplification of a 420-bp gene fragment with 97% sequence homology to the major capsid protein gene of Chilo iridescent virus. These data indicate that this new isolate, which we propose to be termed Gryllus bimaculatus iridescent virus, belongs to the genus Iridovirus of the family Iridoviridae.     

Selection of light or darkness, locomotor, and stridulatory activities in the cricket, Gryllus bimaculatus Degeer (Orthoptera: Gryllidae)

Male crickets, Gryllus bimaculatus Degeer, turned lights on or off in a chamber by a seesaw device: (1) during a 12 h, and (2) during a 24 h day. The crickets in (1) and the last-instar nymph in (2) turned the lights on and off at irregular intervals and duration. The selection rate for darkness was greater than that for light by an average exceeding 80%. The locomotor activity of the nymph in (1) was arrhythmic. In (1) the adults stridulated and were active in continuous darkness during the 12 h, while, in contrast in (2) they turned on the light and stridulated without switching the light off. The locomotor and stridulatory activities of the adult crickets in (2) were free-running. These activities resulted in a free-running rhythm of selection for light or darkness in (2). Under the conditions of the present experiments, the circadian pacemaker functioned in the same way in light and dark cycles as in constant light conditions.     

Secretion of beta-glycosidase by middle midgut cells and its recycling in the midgut of Tenebrio molitor larvae

There are four β-glycosidases (βgly1, βgly2, βgly3, and βgly4) in Tenebrio molitor midgut larvae. βgly1 and βgly2 have identical kinetic properties, and differ in a few amino acid residues. Purified βgly1 was used to raise antibodies in a rabbit. The resulting antiserum recognizes in a Western blot only βgly1 and βgly2 in midgut tissue homogenates and contents. An immunocytochemical study carried out using confocal fluorescence and immunogold techniques showed that βgly1+βgly2 are secreted by exocytosis mainly from the distal part of the second third of T. molitor midguts. This is the first immunocytochemical study of an insect digestive enzyme that does not have polymers as substrates. Enzyme assays with 0.3 mM amygdalin, a condition that detects only βgly1+βgly2, revealed that most of those β-glycosidases are found in the lumen of anterior and middle midgut. This supports the hypothesis that a countercurrent flux of fluid occurs in T. molitor midgut that is able to carry βgly1 and βgly2 to anterior midgut, in agreement with the enzyme recycling mechanism thought to occur in most insects.     

Ontogeny of the cricket Phaeophilacris bredoides Kaltenbach (Orthoptera, Gryllidae)

Development of Phaeophilacris bredoides Kalt. was studied under stable laboratory conditions: temperature of 26°C, air humidity 60%, and 12L: 12D photoperiod (Knyazev, 1985). The life cycle of Ph. bredoides includes four stages: egg, pronymph, nymph, and adult. The duration of embryonic development is 28 days. The nymphal ontogeny lasts 230 days and consists of 25 instars. The duration of nymphal instars (days) is: 1st—16, 2nd—6, 3rd—8, 4th—10, 5th—10, 6th—15, 7th—10, 8th—8, 9th—9, 10th—11, 11th—11, 12th—8, 13th—9, 14th—10, 15th—7, 16th—9, 17th—7, 18th—7, 19th—7, 20th—10, 21st—11, 22nd—7, 23rd—9, 24th—6, and 25th—9. The duration of adult life is 126 days in males and 125 days in females. Three periods were distinguished in the imaginal ontogeny of males and females: pre-reproductive, reproductive, and post-reproductive. The pre-reproductive period begins with the molt to the adult and ends with the onset of reproductive behavior in males and with the first copulation in females. Its duration is 4 (3–6) days in males and 5 (2–7) days in females. The reproductive period in males starts with the onset of reproductive behavior on the 4th (3rd–6th) day and lasts 119 (98–135) days. In females it begins when they start responding to males’ courtship behavior and lasts 116 (97–133) days. The female reproductive period includes two alternating phases: copulation and egg-laying. The egg-laying phase is initiated by successful copulation. The post-reproductive period in females starts when oviposition ceases and in males, when their reproductive behavior disappears. This period lasts about 3 (2–3) days in males and 4 (2–7) days in females, until the insect dies.     

The Energetics of Male Mating Strategies in Field Crickets (Orthoptera: Gryllinae: Gryllidae)

Male field crickets (Gryllinae: Gryllidae: Orthoptera) vary in their mating strategies, particularly in whether they call from defended sites to attract phonotactic females or roam silently in search of potential mates. To better understand the economics underlying these alternative strategies, respirometry was employed to examine the energetic costs of each strategy's component behaviors for a representative species, Acheta domesticus. Advertisement calling in this species, composed of low pulse rate chirps, is an order of magnitude less energetically costly than walking per unit time. However, for gryllids that advertise call with higher pulse rate trills, calling and walking appear to be of equivalent energetic cost. Thus, if energetic costs are important in determining grylline mating strategies, trillers and chirpers should have different sensitivities to change in factors affecting the relative payoffs of each strategy. Field studies of changes in mating behavior with increased population density support this argument. This study also determined that courtship calling by A. domesticus was over twice as energetically costly as advertisement calling per unit time, suggesting its importance as a more reliable indicator of signaler, or mate, quality.     

A taxonomic study of genus Teleogryllus from East Asia (Insecta: Orthoptera: Gryllidae)

Crickets belonging to Teleogryllus spp. are widely distributed in East Asia and they are difficult to distinguish. In this study, a phylogenetic tree of Teleogryllus is reconstructed by COI gene, and all species can be distinguished well. The possible evolutionary histories of male genitalia, songs and life history are discussed. The result supports establishing subgenera Macroteleogryllus and Brachyteleogryllus of Teleogryllus spp. from East Asia. T. boninensis is moved to Brachyteleogryllus. The calling song of four species includes both long and short chirps, and the rest just have single chirps. The species inhabit sympatrically trend to have different type of calling song. Egg overwinter type evolved several times independently.     

Learning and solar orientation in the mole cricket Gryllotalpa gryllotalpa (Orthoptera: Gryllidae)

The ecologically efficient escape direction assumed by Gryllotalpa gryllotalpais learned by the larvae after they leave the maternal nest. Nonetheless, larvae hatched in the laboratory are innately capable of using the sun to maintain a set escape direction. The species can also modify the set point of its sun compass to conform to topographical changes encountered during its wanderings.     

Acoustic communication in the trilling field cricket,Gryllus rubens (Orthoptera: Gryllidae)

Male field crickets produce calling songs that are used for attracting conspecific females for mating. Acoustic communication was studied in the trilling field cricket, Gryllus rubens.A trilled calling song consists of a continuous train of sound pulses, each of which was repeated at a stereotyped rate. Singing males were recorded at different temperatures. The pulse period of the calling song decreased with increasing temperature; there was less effect of temperature on pulse duration. Female phonotaxis was studied on a noncompensating, spherical treadmill. In two-stimulus (choice) playback experiments, females preferred the conspecific trilled song over the chirped calling song of a sympatric species, Gryllus fultoni.This preference persisted even when the song of G. fultoniwas 6 dB louder. Females also discriminated between synthetic trills having different pulse periods; females chose trills with the conspecific pulse period over trills having lower and higher pulse periods.