Flightless Bug Research Articles

Discovery and characterization of a novel alphavirus-like RNA virus from the red firebug Pyrrhocoris apterus L. (Heteroptera)

Recent studies have shown that insects harbor numerous viruses of various taxa and that viral infections are often latent without noticeable symptoms. The red firebug Pyrrhocoris apterus, a true flightless bug from the family Pyrrhocoridae, is widely used for physiological studies on insect metabolism, endocrinology, and digestion. While exploring the transcriptome of P. apterus salivary glands, a nearly complete genomic sequence of a novel RNA virus was reconstructed. The virus, provisionally named Pyrrhocoris apterus virus 1 (PaV1), possesses eight potential open reading frames (ORFs) encoding for an array of proteins, some of which are involved in virus replication while others ensure success of the virus in multiple ways, including evasion of the host immune response. In addition to the information obtained from sequence analyses, we documented virus transmission, virus-induced mortality, host response upon persistent PaV1 infection, virion morphology, and putative virus-induced structures in salivary gland cells in a laboratory culture of red firebug. We propose that PaV1 belongs to a novel viral species of a new, yet-to-be established family.

Regulation of the development of flight muscles in long-winged adults of the flightless bug Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

We studied the development of the indirect flight muscles and reproductive organs in long-winged (macropterous) adults of the flightless bug Pyrrhocoris apterus (L.) and the factors involved in flight muscles histolysis by means of total protein analysis. Both the extirpation of the corpus allatum, an endocrine gland that is the sole source of juvenile hormone, and sham operation accelerated histolysis and decreased the level of the total protein content of the flight muscles to the same extent. Degeneration of flight muscles was not a result of allatectomy but rather a consequence of injury, followed by resumption of enhanced food intake, because it was stimulated also by the removal of wings. Transfer of penultimate instar larvae to a diapause-inducing short-day photoperiod did not prevent imaginal growth and histolysis of flight muscles, but inhibited growth of ovaries in females and maturation of accessory glands in adult males. Thus inactivation of the corpus allatum in diapausing macropters does not prevent imaginal growth of their flight muscles. Application of a high dose of methoprene to the surface of intact long-day macropterous adults induced precocious histolysis of flight muscles and growth of ovaries in females and accessory glands in males. Prolonged starvation of macropterous adults had only a small effect on the histolysis of their flight muscles. The results indicate that imaginal growth and histolysis of indirect flight muscles in macropterous individuals of P. apterus are largely juvenile hormone-independent processes that are programmed to occur spontaneously, but can be affected by various internal and external factors.

Wing morph- and age-related differences in fertilization success of adult males of a flightless bug, Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

The aim of the present study was to test the hypothesis that short-winged (brachypterous) and long-winged (macropterous) adult males of a flightless bug Pyrrhocoris apterus differ in fertilization success. For this purpose, 5, 10 and 28 days old brachyp- terous and macropterous males were mated for the same period of time with reproductively active 5 days old brachypterous females. The average hatchability of five egg batches of these females was used as a measure of the fertilization success of the males. The results revealed significantly higher hatchability of the eggs laid by females that copulated with 5 or 10 days old brachypterous males than with same-aged macropterous males. In contrast, the average hatchability of eggs of females that copulated with 28 days old brachypterous males was significantly lower than of those mated with same-aged macropterous males. It is the first report of wing morph- and age-related differences in fertilization success of males in insects with a non-functional wing-polymorphism. The possible relationship between wing- and age-related differential fertilization and the mating success of P. apterus males, and differ- ences in the amount of secretion transferred from their accessory glands into the reproductive tract of females during copulation, is discussed.

Crustacean red pigment-concentrating hormone Panbo-RPCH affects lipid mobilization and walking activity in a flightless bug Pyrrhocoris apterus (Heteroptera) similarly to its own AKH-peptides

In the present study we tested whether the walking activity of macropterous females of the flightless wing-polymorphic bug Pyrrhocoris apterus (L.) can be stimulated by its native adipokinetic hormone Peram-CAH-II and the crustacean red pigment-concentrating hormone (Panbo-RPCH), and the effectiveness of the latter hormone in a lipid mobilization assay. Two different doses (10 or 40 pmol) of Peram-CAH-II or Panbo-RCPH were injected into 10-day-old macropterous females of P. apterus to evaluate their effects on the walking activity of treated females. The results obtained showed a significant stimulation of walking activity only with the lower dose (10 pmol) of either hormone Peram-CAH-II or Panbo-RPCH. On the contrary, the walking activity of the same-aged females of macropterous morph treated with the higher dose (40 pmol) of these hormones was decreased. The energy substrates mobilized in Panbo-RPCH-treated macropterous females were lipids. The question of whether the stimulation of locomotion by Panbo-RPCH is limited only to P. apterus or if it might also represent an important function of this hormone in other insects or even in crustaceans is discussed.

Factors terminating ovarian arrest in long-winged females of a flightless bug, Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

The aim of the present study was to determine the factors that are involved in termination of a non-diapause type of ovarian arrest in the adult macropterous females of a flightless bug, Pyrrhocoris apterus (L.), reared under a long-day (18L : 6D) photoperiod. Application of an active analogue of juvenile hormone, methoprene, to adult macropterous females kept under the above conditions induced precocious termination of ovarian arrest and shortened the length of the pre-oviposition period. The results indicate that a temporary ovarian arrest in spontaneously fasting long-day macropterous females results from a deficiency of juvenile hormone. The length of the pre-oviposition period was shortened and ovarian arrest terminated also by de-alation, high temperature and by a prolonged period of starvation. Mating of long-day macropterous females with reproductively active males had no effect on the length of the pre-oviposition period. There was a relationship between the length of the starvation period and the post-feeding pre-oviposition period. The longer the starvation period, the shorter the period from when food was supplied to first oviposition. The results indicate that depletion of the fat body reserves resulting from prolonged fasting, followed by resumption of food intake are pre-requisites for full activation of the corpus allatum and egg development, and play a role in completion and termination of non-diapause ovarian arrest in long-day macropterous females. This phenomenon was never observed in short-day brachypterous females in reproductive diapause.

Endocrine regulation of the reproductive arrest in the long-winged females of a flightless bug, Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

Mechanisms of the suppression of gonadotropic activity of the corpus allatum (CA) in females were com- pared with those previously reported for either or non-diapause females by reciprocal transplanta- tions of the neuroendocrine complexes (comprising the brain-suboesophageal ganglion-corpora cardiaca-CA). The denervated CA stimulated reproduction in most females of all experimental groups suggesting an inhibition of the CA via nervous connections with the brain. The inhibition of the CA within the transplanted neuroendocrine complex was measured by the reproductive performance of feeding recipient females deprived of their own CA. The complex from non-diapause females stimulated reproduction in 58.3-78.9% of recipients suggesting that the inhibition of the CA was mostly overcome by the stimulating internal milieu of feeding females. In contrast, the macropterous complex stimulated reproduction in only 18.8-37.5% of recipients, similar to the diapause brachypterous complex (32.0%). The results indicate that the macropterism, similar to the diapause, is associated with a considerably lower responsiveness of the neuroendocrine complex to humoral stimulation by feeding compared to the responsivenes of the starving complex from non-diapause females. On the other hand, the CA of females is of intermediate size between that of the feeding non-diapause and females, similar to the CA of the non-diapause females deprived of food. Overall, the data suggest that the suppression of the CA activity results from a combination of the diapause-like refractoriness of the neuroendocrine complex with the starvation-like inhibition of the CA growth. Regulation of the CA activity is discussed in relation to the oogenesis-flight syndrome recorded for flying wing- polymorphic species of insects.

Corpus allatum volume-dependent differences in accessory gland maturation in long- and short-winged males of Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

In the present study the temporal changes in the volume of the corpus allatum in three experimental groups of adult males (macropterous, reproductive brachypterous and diapausing brachypterous) of the flightless bug Pyrrhocoris apterus were determined and related to the size of male accessory glands. The results revealed wing morph- and age-dependent differences in the corpus allatum volume in males of this bug. In 4-14 day old males, the volumes of the corpus allatum and accessory glands were largest in long-day reproductive brachypters, intermediate in long-day macropters, and smallest in short-day diapausing brachypters. The smaller corpus allatum in young macropterous males than in same aged reproductive brachypterous males was due to the sponta- neous fasting of the former. Later, starting on day 18 after adult emergence, i.e. when macropterous males were feeding normally, there were no significant differences in the volumes of the corpus allatum between long-day brachypterous and macropterous males. On the other hand, the corpus allatum of 18-28 day old diapausing brachypterous males was significantly smaller than that of same aged long-day macropterous and reproductive brachypterous males. The sizes of the corpus allatum and accessory glands were sig- nificantly positively correlated in macropterous and diapausing brachypterous males. This is the first report of corpus allatum volume-dependent wing morph-related differences in the rate of accessory gland maturation in males of insects with a non-functional macropterism. The role of differential activity of the corpus allatum in the different life history strategies of males of the two wing morphs in this wing-polymorphic insect is discussed.

Flight muscles polymorphism in a flightless bug, Pyrrhocoris apterus (L.): Developmental pattern, biochemical profile and endocrine control

The flightless bug Pyrrhocoris apterus (L.) is polymorphic for both wing length and flight muscle development. The developed flight muscles of macropterous adults of both sexes first enlarge their volume during the first 5 days after adult emergence, but are then histolyzed in all males and females older than 10 and 14 days, respectively. The flight muscles of brachypterous adult males and females are underdeveloped due to their arrested growth. The total protein content of histolyzed dorsolongitudinal flight muscles from 21-day-old macropterous adults of both sexes is lower than that of developed dorsolongitudinal flight muscles in 5–10-days-old macropterous bugs, but substantially higher than the protein content of underdeveloped dorsolongitudinal flight muscles from adult brachypters. Histolyzed dorsolongitudinal flight muscles differ from the developed ones by decreased quantities of 18 electrophoretically separated proteins. Histolysis of developed dorsolongitudinal flight muscles is accompanied by significant decreases in citrate synthase, glyceraldehyde-3-phosphate dehydrogenase and β-hydroxyacyl-CoA dehydrogenase enzyme activities and an increase in alanine aminotransferase activity, and can be precociously induced by application of a juvenile hormone analogue. This is the first report of flight muscle polymorphism, histolysis of developed flight muscles and its endocrine control in insects displaying non-functional wing polymorphism.

Wing morph-specific differences in the metabolism and endocrine control of reserve mobilization in adult males of a flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

The differences in the metabolism and endocrine control of reserve mobilization in long-winged (macropterous) and short-winged (brachypterous) males of a flightless firebug (Pyrrhocoris apterus) were studied. We found that protein content in the gut was significantly lower in 5-10 day-old macropterous males due to their fasting and higher in 28 day-old ones than in the same aged brachypterous counterparts as the result of renewed food intake. Overall protease activity was significantly lower in 10-14 day-old macropters, while an abrupt increase in the activity starting on day 21 after adult ecdysis was also associated with renewal of the food intake. The levels of carbohydrates in haemolymph were only slightly lower in 1-10 day-old macropterous males than in the same aged brachypters. However, more than twofold higher lipid content in haemolymph of 7-10 day-old macropterous males than in the same aged brachypterous males was found. Higher mobilization of lipid reserves from the fat bodies in macropterous males was accompanied by more intensive adipokinetic response and higher levels of adipokinetic hormone in the body. It is the first report of endocrine regulation of wing morph-related differences in the lipid mobilization in males of wing-polymorphic insects.

Wing morph-related differences in the pattern of mating activity in a flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

Summary The mating activity of the long-winged (macropterous) and short-winged (brachypterous) adult males and females from a temperate population (Czech Republic) of the firebug Pyrrhocoris apterus (L.) was studied during the first 14 days of adult life under constant laboratory conditions. The study showed that macropterous pairs copulated almost three times less frequently than the brachypterous pairs. The mean inter-copulation period was significantly longer in macropterous females than in brachypterous ones. Despite the fact that there was no significant difference between the total courtship frequencies of macropterous and brachypterous males, the mean duration of the inter-mating period was significantly shorter in brachypterous males than in macropterous ones, which indicates higher mating vigour of the former. Thus, the lower mating propensity of macropterous pairs was mainly due to lowered receptivity of females and in some extent to lowered mating vigour of males. The courtships of males and copulations of pairs of both wing morphs occurred predominantly throughout the photophase. The mean (peak) time of courtship and copulation activities of brachypterous pairs occurred at about 17:30 MET and 19:00 MET before the onset of the scotophase, respectively. In macropterous pairs the mean peak time of courtship and copulation occurred 2 h earlier. The study showed that the wing morph-related differences in mating activity characteristic for many flying wing-polymorphic insects are maintained also in insects whose all macropterous specimens have evolved flightlessness.

Mating behaviour and wing morph-related differences in the sexual activity of a flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

The sexual activity of long-winged (macropterous) and short-winged (brachypterous) adult males and females of Pyrrhocoris apterus (L.) from a temperate opulation (Czech Republic) were compared. The long-term arena tests revealed postponed sexual maturation and lower sexual activity (longer precopulatory period, longer inter-copulatory period, lower copulation frequency and shorter total duration of copulations) of macropterous pairs when compared with brachypterous pairs of the same age. The total sexual activity of both macropterous and brachypterous pairs increased with the age of the adults, but was significantly lower in the macropterous pairs. The short-term tests showed that the lowered mating propensity of macropterous pairs was mainly due to a decrease in the sexual receptivity of the females. It is the first demonstration of wing morph-related differences in the mating activity of P. apterus females and a first report of a mating disadvantage in the macropterous morph of a wingpolymorphic insect whose macropterous specimens have evolved flightlessness. Evolutionary transformation of the “oogenesis-flight” syndrome, that occurs in flying insects, into an “oogenesis-walking” syndrome in the flightless macropters of P. apterus is discussed.

Wing morph‐related differences in the walking pattern and dispersal in a flightless bug, Pyrrhocoris apterus (Heteroptera)

We tested the hypothesis that adults of flightless long‐winged (macropterous) and short‐winged (brachypterous) morphs of Pyrrhocoris apterus (L.) (Heteroptera) differ in their walking path characteristics and dispersal ability. Descriptive variables such as the distance moved, mean velocity, time in observation zone, turn angle, angular velocity, meander and sinuosity were computed from walking track data obtained with the use of the computerized system EthoVision. The macropterous bugs differed from the brachypterous ones by higher mean and angular velocity and distance moved, by shorter time spent in the observation zone, and by lower turn angle, meander and sinuosity parameters. The analysis of locomotory tracks revealed that the walking path of macropters is less tortuous than that of brachypters. Estimation of mean spatial displacement distance attained by two wing morphs indicate higher dispersal activity of macropters, and an important role of the macropterous morph in the dispersal strategy of this bug and spreading of the macropterous genotype into new areas. The results obtained are in accordance with our hypothesis that flightless macropters of P. apterus maintain higher dispersal ability than brachypters despite the evolutionary loss of flight capability.

Topical application of Pya‐AKH stimulates lipid mobilization and locomotion in the flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

Abstract Two different methods of applying Pya‐AKH to long‐winged (macropterous) females of the firebug, Pyrrhocoris apterus (Linnaeus) (Heteroptera) were compared: both injection and topical application increased the levels of lipids in the haemolymph and stimulated locomotor activity. Lipid mobilization was maximal when 10 pmol was applied by injection or 40–100 pmol by topical application, with the first significant responses occurring 1.5 h after injection and 2 h after topical application. The highest elevations of lipid concentration in the haemolymph were comparable between the treatments, i.e. 14.36 ± 3.59 mg/mL for injection and 14.43 ± 4.07 mg/mL for topical application. However, these maximal elevations were achieved at different times: 3 h after the injection and 7 h after the topical application.Injection of 10 and 40 pmol of Pya‐AKH stimulated locomotor activity with maximal activity 3 h later but, surprisingly, injection of 80 pmol showed no effect initially and than a slight inhibitory effect after 6–8 h.Increased locomotor activity was found after topical application of Pya‐AKH, but the response was lower than after injection and appeared later, 5–9 h after the hormone application. In addition, the greatest increase in walking activity required topical application of 300 pmol and was still less dramatic than the response to injection. The stimulatory effect of Pya‐AKH on locomotion was positively correlated with its effect on lipid mobilization only for injection of the hormone. It is argued that a stress caused by injection could play a role in the appearance of the complex response to adipokinetic hormone.

Latitudinal gradient in response of wing polymorphism to photoperiod in a flightless bug, Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

The aim of this study was to determine if there is a latitudinal gradient in the photoperiodic regulation of wing dimorphism in the flightless bug, Pyrrhocoris apterus (L.). For this purpose individuals from three geographical populations (Israel, Spain and Czech Republic) were reared under different photoperiods and the wing length of the adults analyzed. The highest percentage of long-winged (macropterous) specimens was found in the population from Israel (35.3%), whereas percentages of macroptery were lower in the cultures from Spain (9.5%) and Czech Republic (8.6%). A higher proportion of macropterous specimens was recorded in the northern population of P. apterus kept under long daylengths (Czech Republic, 16 h) than in the southern populations (Spain, 14-15 h; Israel, 12 h). The results indicate that there is a latitudinal gradient in the critical photoperiod determining wing length in P. apterus.

Locomotor activity in adult Pyrrhocoris apterus (Heteroptera) in relation to sex, physiological status and wing dimorphism

Summary The pattern of locomotor (walking) activity was studied in adult males and females of short-winged (brachypterous) and long-winged (macropterous) morph of the flightless bug Pyrrhocoris apterus (Linnaeus) (Heteroptera: Pyrrhocoridae) under constant laboratory conditions. Walking activity was measured with a computerized video system and analysed with respect to sex, physiological status (reproduction, diapause and reproductive arrest of non-diapause type) and wing dimorphism of the bugs. The largest duration was observed in the macropterous females with reproductive arrest of non-diapause type (average 6 h per day) and the shortest duration in diapausing brachypterous females and males (average less than 2 h per day). This was reflected also in the overall time spent by walking during the first 14 days after imaginal ecdysis. The time spent by walking significantly increased in the macropterous morph as the bugs aged, whereas in diapausing brachypters the time spent by walking decreased with age. No linear relationship between walking activity and age was found in reproductive brachypterous morph. The bugs of all experimental groups moved mostly during the photophase and were almost inactive during the scotophase. Thus, walking activity in P. apterus is diurnal, irrespective of the wing morph, physiological status, sex and age. Contrary to the macropterous morph, where the locomotor activity of females during photophase was significantly higher than in males, no significant differences were found between the locomotor activities of brachypterous males and females. The observed differences in locomotor activity are discussed in relation to different roles of two wing morphs in the life history of this heteropteran.

Wing movement behavior in long- and short-winged morphs of the flightless bug Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae)

The aim of the present paper was to study, with the use of video-recordering and computerized methods, the flight and wing movement behavior in two wing morphs of the flightless bug Pyrrhocoris apterus during the first 14 days of their adult life. We demonstrated the capability of macropterous males and females of this heteropteran to flap their fore- and hindwings for the first time. Flight was not observed in any of the tested males and females. We revealed that wing flapping does not occur earlier than on the fourth and fifth day after adult emergence in males and females, respectively. Time-series analysis of the obtained data showed that the maximum activity of wing flaps is limited to the light phase of the 12-h light/12-h dark photocycle, with the peak occurring in the morning, i.e., about 3 h after turning the light on. No substantial differences in the peaks of wing flapping activities in males versus females were found. The present paper is the first experimental demonstration of wing flap activity in a representative species with a “nonfunctional” alary polymorphism.