How-Jing Lee

Proteome mining for novel IgE‐binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE‐mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2‐DE and IgE‐binding proteins were analyzed by nanoLC‐MS/MS or N‐terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE‐binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross‐reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE‐binding proteins varied between cockroach‐allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.

Pigment dispersing factor: An output regulator of the circadian clock in the German cockroach

Pigment-dispersing factor (PDF) is a neuropeptide that is synthesized specifically and constantly in the circadian clock cells of many insects. The functions of PDF have not been fully determined, but it might serve as the output and coupling signal of circadian locomotor rhythms. In this experiment, we explore the functions of PDF in the German cockroach with RNA interference technique. Since the 2nd day after pdf double-strand RNA (dsRNA) injection, the amount of pdf mRNA decreased significantly, and this knockdown effect could persist at least 56 days. With immunostaining technique, the clock cells of pdf dsRNA-injected cockroaches could not be stained by anti-PDF antibody. In the behavioral study, pdf dsRNA injection caused rhythmic males to become arrhythmic in light-dark cycles or in constant darkness. In addition, due to the nocturnal nature of the German cockroaches, the locomotor activity increased after lights-off or entering subjective night. However, this activity peak gradually disappeared after pdf dsRNA injection. Based on these 2 lines of evidences, PDF serves as an output regulator of locomotor circadian rhythm in the German cockroach.

Parallel control mechanisms underlying locomotor activity and sexual receptivity of the female German cockroach, Blattella germanica (L.)

We have studied the effects of ovaries, juvenile hormone (JH) and mating on locomotor activity and sexual receptivity of female German cockroaches. Our results indicate that locomotor activity and sexual receptivity are under the same control mechanisms. The ovary served as a negative masking factor for the locomotor circadian rhythm, but did not affect the frequency of locomotor activity. We conclude that JH controls the locomotor activity of females from the following evidence: (1) increasing locomotion of virgin females coincided with an increasing volume of the corpora allata; (2) allatectomy reduced female locomotion significantly; (3) after absorbing the JH analogue (fenoxycarb) through their tarsi, allatectomized females regained their high level of locomotor activity. Since the daily locomotor activity of allatectomized and ovari-allatectomized females changed cyclically with continuous (non-cyclic) contact of fenoxycarb, an unidentified factor which was independent of ovarian development is proposed to regulate cyclic locomotor activity. In addition to controlling the frequency of locomotor activity, JH was essential for the expression of the locomotor circadian rhythm because allatectomy abolished the circadian rhythm expressed in ovariectomized females. Mating significantly decreased the frequency of locomotor activity and the degree of sexual receptivity. The inhibitory effect of mating resulted from the transmission of a mating signal through the ventral nerve cord when sperm was transferred successfully. The mating experiments with allatectomized and ovariectomized females showed that JH was the major factor in regulating the expression of sexual receptivity.

Mating effects on the feeding and locomotion of the German cockroach, Blattella germanica

Abstract. Feeding cycles and daily locomotory patterns of the German cockroach, Blattella germanica L. (Dictyoptera: Blattellidae), were correlated with the ovarian development cycle. To meet the nutrient requirement for ovarian development, females increased feeding before forming oothecae. Locomotory activity also increased when females became sexually receptive. All these activities reached a peak just before the formation of oothecae. Ovarian development ceased and locomotion and food consumption decreased during pregnancy. Both mated and virgin females showed similar reproductive cycles, but those of mated females were more precisely timed (intervals between successive oothecae, and pregnancy duration, were 5 ± 0.6 and 17 ± 0.6 days, respectively). However, the intervals between successive oothecae of virgin females were longer and less synchronized. During this longer interval, feeding took place immediately following the discharge of the ootheca, but locomotory activity increased 5 days later when females became sexually receptive. Mated females increased locomotory activities 1 or 2 days before the end of pregnancy, presumably searching for deposition sites for oothecae. Female adults were found to exhibit a daily nocturnal locomotory pattern. However, under the physiological demands of reproduction, the pattern could be changed, for example by increasing activity during photophase when females were sexually receptive. The physiological effects of reproduction override the control of the daily locomotory pattern by its diel clock.

Mapping the cellular network of the circadian clock in two cockroach species

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.

Cryptochrome 2 mediates directional magnetoreception in cockroaches

Significance The photosensitive protein Cryptochrome (Cry) is involved in the detection of magnetic fields (MFs) in Drosophila. However, Cry-dependent responses to natural MF intensities and to the direction of the MF vector have not been demonstrated previously in any insect. Birds, monarch butterflies, and many other species perceive the direction of geomagnetic field (GMF) lines, but the involvement of Cry has not been rigorously proven using genetic tools. In this study, by combining behavioral and genetic approaches, we provide the first unambiguous evidence to our knowledge of a Cry-dependent sensitivity to the direction of GMF in two cockroach species. Furthermore, by eye-covering experiments and by immunolocalization of a crucial mammalian-type Cry2 under the retina, we clearly show that the eye is an indispensable organ for the directional GMF response. The ability to perceive geomagnetic fields (GMFs) represents a fascinating biological phenomenon. Studies on transgenic flies have provided evidence that photosensitive Cryptochromes (Cry) are involved in the response to magnetic fields (MFs). However, none of the studies tackled the problem of whether the Cry-dependent magnetosensitivity is coupled to the sole MF presence or to the direction of MF vector. In this study, we used gene silencing and a directional MF to show that mammalian-like Cry2 is necessary for a genuine directional response to periodic rotations of the GMF vector in two insect species. Longer wavelengths of light required higher photon fluxes for a detectable behavioral response, and a sharp detection border was present in the cyan/green spectral region. Both observations are consistent with involvement of the FADox, FAD•− and FADH– redox forms of flavin. The response was lost upon covering the eyes, demonstrating that the signal is perceived in the eye region. Immunohistochemical staining detected Cry2 in the hemispherical layer of laminal glia cells underneath the retina. Together, these findings identified the eye-localized Cry2 as an indispensable component and a likely photoreceptor of the directional GMF response. Our study is thus a clear step forward in deciphering the in vivo effects of GMF and supports the interaction of underlying mechanism with the visual system.

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.

Circadian control of permethrin-resistance in the mosquito Aedes aegypti.

Daily fluctuation of permethrin-resistance was found in adult mosquito Aedes aegypti, the major vector of dengue viruses in Taiwan. We hypothesized there is a relationship between resistance and the circadian clock. To test our hypothesis we correlated changes in the knock-down time (KT(50)) response to permethrin with the expression of the pyrethroid-resistant gene CYP9M9 and the clock gene period (per) during a 12:12h photoperiodic cycle. Rhythmic expression of per peaked at early scotophase of the light-dark cycle and at early subjective night in constant darkness. The values of KT(50) and the expression of CYP9M9 also exhibited circadian rhythms in both susceptible and permethrin-resistant mosquito strains, from which we inferred a link to the circadian clock. The KT(50) was significantly longer in the light than in the dark phase, and the level of CYP9M9 mRNA was maximal in early scotophase, dropped to a minimum in the midnight and then slowly increased through the photophase. Existence of a clock control over mosquito sensitivity to permethrin was further indicated by reduced expression of CYP9M9 and reduced mosquito resistance to permethrin after temporal silencing of the per gene. These data provide the first evidence on the circadian control of insect resistance to permethrin.

Adipokinetic hormone counteracts oxidative stress elicited in insects by hydrogen peroxide: in vivo and in vitro study

The role of adipokinetic hormone (AKH) in counteracting oxidative stress elicited in the insect body is studied in response to exogenously applied hydrogen peroxide, an important metabolite of oxidative processes. In vivo experiments reveal that the injection of hydrogen peroxide (8 µmol) into the haemocoel of the firebug, Pyrrhocoris apterus L. (Heteroptera: Pyrrhocoridae) increases the level of AKH by 2.8‐fold in the central nervous system (CNS) and by 3.8‐fold in the haemolymph. The injection of hydrogen peroxide also increases the mortality of experimental insects, whereas co‐injection of hydrogen peroxide with Pyrap‐AKH (40 pmol) reduces mortality to almost control levels. Importantly, an increase in haemolymph protein carbonyl levels (i.e. an oxidative stress biomarker) elicited by hydrogen peroxide is decreased by 3.6‐fold to control levels when hydrogen peroxide is co‐injected with Pyrap‐AKH. Similar results are obtained using in vitro experiments. Oxidative stress biomarkers such as malondialdehyde and protein carbonyls are significantly enhanced upon exposure of the isolated CNS to hydrogen peroxide in vitro, whereas co‐treatment of the CNS with hydrogen peroxide and Pyrap‐AKH reduces levels significantly. Moreover, a marked decrease in catalase activity compared with controls is recorded when the CNS is incubated with hydrogen peroxide. Incubation of the CNS with hydrogen peroxide and Pyrap‐AKH together curbs the negative effect on catalase activity. Taken together, the results of the present study provide strong support for the recently published data on the feedback regulation between oxidative stressors and AKH action, and implicate AKH in counteracting oxidative stress. The in vitro experiments should facilitate research on the mode of action of AKH in relation to oxidative stress, and could help clarify the key pathways involved in this process.

The expression of locomotor circadian rhythm in female German cockroach, Blattella germanica (L.).

Fifteen percent of intact female German cockroaches (n = 13), Blattella germanica (L.) (Dictyoptera: Blattellidae), had weak free-running locomotor rhythmicity under 28 degrees C and constant darkness conditions. However, 86% of ovariectomized females (n = 14) showed a strong free-running rhythm under the same conditions with a circadian period of 23.60 +/- 0.15 h, similar to the males period of 23.45 +/- 0.03 h. In addition, the locomotory activities occurred mainly during the subjective night under DD conditions as was the case in males. These results indicated that female locomotion was under the control of a circadian oscillator, which was masked by the existence of ovaries. This internal masking effect could be removed by the existence of males, but females had no effect on the locomotor pattern of another female. Since the male failed to entrain female locomotion, its role as a zeitgeber was excluded. That the locomotory pattern of the females still coincided with their reproductive cycle when exposed to male odor suggests that exposure to a male only partially removed the internal masking effects.