Larval-pupal Ecdysis Research Articles

Parasitism by the Tachinid Parasitoid Exorista japonica Leads to Suppression of Basal Metabolism and Activation of Immune Response in the Host Bombyx mori.

Simple SummaryThe dipteran parasitoid Tachinidae are important biocontrol agents and some of them are pests in sericulture. We previously have demonstrated that tachinid parasitoid Exorista japonica parasitism causes pupation defects in Bombyx mori. However, the underlying mechanism is not fully understood. In this study, we performed transcriptome analysis of the fat body of B. mori parasitized by E. japonica. We found that the host basal metabolism was inhibited whereas the immune response was activated. These results indicate that the tachinid parasitoid perturbs the basal metabolism and activates the energetically costly immunity of the host, leading to the development arrest of the host. This study provides insights into how tachinid parasitoids modify host basal metabolism and immune response for the benefit of developing parasitoid larvae.The dipteran tachinid parasitoids are important biocontrol agents, and they must survive the harsh environment and rely on the resources of the host insect to complete their larval stage. We have previously demonstrated that the parasitism by the tachinid parasitoid Exorista japonica, a pest of the silkworm, causes pupation defects in Bombyx mori. However, the underlying mechanism is not fully understood. Here, we performed transcriptome analysis of the fat body of B. mori parasitized by E. japonica. We identified 1361 differentially expressed genes, with 394 genes up-regulated and 967 genes down-regulated. The up-regulated genes were mainly associated with immune response, endocrine system and signal transduction, whereas the genes related to basal metabolism, including energy metabolism, transport and catabolism, lipid metabolism, amino acid metabolism and carbohydrate metabolism were down-regulated, indicating that the host appeared to be in poor nutritional status but active in immune response. Moreover, by time-course gene expression analysis we found that genes related to amino acid synthesis, protein degradation and lipid metabolism in B. mori at later parasitization stages were inhibited. Antimicrobial peptides including Cecropin A, Gloverin and Moricin, and an immulectin, CTL11, were induced. These results indicate that the tachinid parasitoid perturbs the basal metabolism and induces the energetically costly immunity of the host, and thus leading to incomplete larval–pupal ecdysis of the host. This study provided insights into how tachinid parasitoids modify host basal metabolism and immune response for the benefit of developing parasitoid larvae.

Developmental inhibitory effect of the Syzygium aromaticum essential oil on the postembryonic stages of a polyphagous pest, Pericallia ricini (Lepidoptera: Arctiidae)

ABSTRACT The chemical composition of Syzygium aromaticum essential oil (EO), and its efficacy on the development and metamorphosis of polyphagous pest, Pericallia ricini was assessed. The chemical composition of the S. aromaticum EO was analysed by gas chromatography and mass spectrometry method. The major volatile components present in the EO were eugenol, β-caryophyllene, caryophyllenyl alcohol, α-humulene, clovene, globulol, and eugenyl acetate. For topical bioassay, EO was tested against the sixth (penultimate) and seventh (ultimate) larval instars of P. ricini resulted in the disruption of normal moulting and metamorphosis, leading to various developmental aberrations. The results comprised larval mortality, delayed larval-larval, and larval-pupal ecdysis duration, ecdysial failure, formation of non-viable intermediates, emergence of abnormal adults, reduced pupation, and inhibition of total adult emergence. This study also reports about the juvenoid nature of clove oil for the first time. Our finding demonstrates that S. aromaticum EO has significant negative effects on P. ricini, and suggests that it can be implemented for the successful control of this economically important insect.

Effect of the medicinal plant Withania somnifera on the development of a medico-veterinary pest Chrysomya megacephala (Diptera: Calliphoridae)

ABSTRACT Topical administration of root and seed extracts (5, 10, 15 and 20 µg/µL) of Withania somnifera to 0, 1, 2, and 3 days post-moult third instar larvae of Chrysomya megacephala resulted in disruption of moulting and metamorphosis. This leads to several developmental aberrations, such as prolongation of larval-pupal and pupal-adult ecdysis, larval mortality, abnormal pupariation, formation of pupal-adult mosaics and adultoids, ecdysial failure, reduced pupariation and adult emergence. Treatment with seed extracts of W. somnifera was more effective as it causes greater toxicity in all the treated groups as compared to that observed with the root extracts. There was a significant interaction among all three factors viz. extract type (root and seed extracts), larval stage (0, 1, 2, 3 days post-moult third instar larvae) and concentration (5 µg/µL, 10 µg/µL, 15 µg/µL, 20 µg/µL) for prolongation of larval-pupal and pupal-adult ecdysis. The results are similar to those observed with the administration of juvenoids and hence it can be inferred that the plant extracts mimic the action of juvenoids. The results clearly suggest that W. somnifera acts as a potential IGR disrupting the moulting and metamorphosis as a consequence of interference with the normal hormonal mechanism in C. megacephala. Abbreviations: Co Sf=coarse surface; Cy W=cylindrical wings; De Ae = deformed anterior end; De W = deformed wings; Ev G = everted genitalia; Ev Pt = everted ptilinium; L Mp = larval mouth parts; Nm W = non-membranous wings; P Ab = pupal abdomen; P Cu = pupal cuticle; Pa T L = partial tanned legs; Pg E = pigmented eyes; Pm = puparium; Re W = reduced wings; Sr G = scar of genitalia; T Pm = tanned puparium; Tu L = tubular legs; Tw Pm = twisted puparium; Un Ab = untanned abdomen; Un Pm = untanned puparium; W Pri = wing primordia

Toxicity and insect growth regulatory effects of root extract from the medicinal plant, Withania somnifera (Linnaeus) against red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

The insect growth regulatory effects of the medicinal plant, Withania somnifera (Family: Solanaceae) on the moulting and metamorphosis of economically important stored grain pest, Tribolium castaneum (Coleoptera: Tenebrionidae) were assessed under laboratory conditions. Seventh instar larvae of the red flour beetle, T. castaneum were topically treated with different doses of root extract of W. somnifera. Treatment with root extract of W. somnifera disrupted growth, development, moulting and metamorphosis in T. castaneum. The results included mortality, prolongation of larval-pupal and pupal-adult ecdysis, ecdysial stasis, development of larval-pupal and pupal-adult intermediates and adultoids, eclosion inhibition, reduced pupation and adult emergence. The LC50 value in topical application of root extract of W. somnifera was 0.5850 µg·µL−1 against last instar larvae of T. castaneum. These results are similar to those observed by the application of juvenile hormone analogues and may be due to interference with the normal hormonal mechanism of moulting and metamorphosis.

Importance of Taiman in Larval-Pupal Transition in Leptinotarsa decemlineata.

Insect Taiman (Tai) binds to methoprene-tolerant to form a heterodimeric complex, mediating juvenile hormone (JH) signaling to regulate larval development and to prevent premature metamorphosis. Tai also acts as a steroid receptor coactivator of 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and Ultraspiracle (USP), to control the differentiation of early germline cells and the migration of specific follicle cells and border cells in ovaries in several insect species. In holometabolous insects, however, whether Tai functions as the coactivator of EcR/USP to transduce 20E message during larval-pupal transition is unknown. In the present paper, we found that the LdTai mRNA levels were positively correlated with circulating JH and 20E titers in Leptinotarsa decemlineata; and ingestion of either JH or 20E stimulated the transcription of LdTai. Moreover, RNA interference (RNAi)-aided knockdown of LdTai at the fourth (final) instar stage repressed both JH and 20E signals, inhibited larval growth and shortened larval developing period. The knockdown caused 100% larval lethality due to failure of larval-pupal ecdysis. Under the apolysed larval cuticle, the LdTai RNAi prepupae possessed pupal thorax. In contrast, the process of tracheal ecdysis was uncompleted. Neither JH nor 20E rescued the aforementioned defectives in LdTai RNAi larvae. It appears that Tai mediates both JH and 20E signaling. Our results uncover a link between JH and 20E pathways during metamorphosis in L. decemlineata.

Sensitivity of tobacco caterpillar, Spodoptera litura, to extract from a medicinal plant, Withania somnifera

ABSTRACTThe larval stage of a polyphagous moth Spodoptera litura (Lepidoptera: Noctuidae), commonly known as tobacco caterpillar, causes loss in many vegetables. Use of chemical pesticides to control this pest has resulted in environmental pollution, is hazardous to non-target organisms, and contributes to development of pest resistance against these compounds. Alternatives for pest management need to be developed. Extracts derived from plants can function as insect growth regulators and are easily biodegradable. Topical administration of root extracts from a medicinal plant, Ashwagandha (Withania somnifera L. Dunal), to sixth instar larvae and freshly molted pupae (0–2 h old) of S. litura caused prolongation of larval-pupal and pupal-adult ecdysis duration, mortality, reduced pupation, and adult emergence. Root extracts of W. somnifera produced the morphological and developmental abnormalities like ecdysial failure, formation of larval-pupal and pupal-adult intermediates, abnormal pupae and adultoids. Treatment with root extracts of W. somnifera was more toxic to pupae compared to larvae of S. litura. The results may be due to interference with normal hormonal mechanism eventually leading to disruption of molting and metamorphosis.

RNA interference against the putative insulin receptor substrate gene chico affects metamorphosis in Leptinotarsa decemlineata

It is noted that insect insulin/insulin-like growth factor/target of rapamycin signaling is critical for the regulation of metamorphosis in holometabolous insects. However, the molecular mechanism remains undetermined. Our previous findings reveal that RNA interference (RNAi)-mediated knockdown of an insulin gene (LdILP2) in Leptinotarsa decemlineata disturbs both 20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling, and impairs pupation. In the present paper, we further observed that the expression of the insulin receptor substrate gene chico (Ldchico) and the phosphoinositide-3-kinase gene pi3k (Ldpi3k92E) was repressed in LdILP2 depleted larvae. Moreover, RNAi of Ldchico or Ldpi3k92E decreased food consumption, affected absorption and metabolism of amino acids and sugars, and reduced expression of several 20E (LdEcR, LdHR3 and LdE75) and JH (LdJHAMT, LdKr-h1 and LdHairy) signaling genes. As a result, larval development was postponed and larval growth was inhibited. Intriguingly, knockdown of Ldchico, rather than Ldpi3k92E, impaired larval-pupal and pupal-adult ecdysis, and specifically repressed transcription of another 20E signaling gene LdUSP. Ingestion of 20E rescued the expression of LdEcR, LdHR3 and LdE75, whereas 20E feeding restored neither the decreased LdUSP mRNA level, nor the reduced pupation and adult emergence rates in Ldchico RNAi larvae. Therefore, Chico is critical for the regulation of larval-pupal-adult transition by a PI3K-independent pathway, perhaps through activation of USP in L. decemlineata.

Effect of Teflubenzuron Ingestion on Larval Performance and Chitin Content in Leptinotarsa decemlineata

The potential of teflubenzuron was assessed in a series of laboratory studies in order to achieve consistent, long-term, integrated management of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Teflubenzuron exhibited excellent stomach toxicity to the larvae. Its larvicidal activity was comparable with those of cyhalothrin, chlorantraniliprole and spinosad. Moreover, the teflubenzuron-fed larvae consumed less foliage, grew slower, and needed a longer period to develop, in a dose dependent manner. Most of these larvae died during larval-larval molting, larval-pupal ecdysis or adult emergence. Furthermore, chitin contents in body carcass (without midgut) and integument specimen of the teflubenzuron-fed larvae significantly decreased, whereas the chitin amount in the midgut peritrophic matrix was not affected. In addition, uridine diphosphate-N-acetylglucosamine-pyrophosphorylase gene (LdUAP1), which was mainly responsible for chitin biosynthesis in ectodermally-derived tissues, was suppressed after teflubenzuron ingestion, in contrast to its partner LdUAP2 for chitin formation in the midgut peritrophic matrix. In a word, by inhibition of chitin production in ectodermally-derived tissues, teflubenzuron is an effective benzoylurea insecticide to L. decemlineata larvae. It can be a valuable tool in effective integrated pest management and insecticide resistance management programs against L. decemlineata.

Withania somnifera acts as a potential insect growth regulator in the polyphagous pest, Pericallia ricini

Abstract Both seed and root extracts of the medicinal plant, Ashwagandha, Withania somnifera exhibit insect growth regulatory activity against the polyphagous pest, Pericallia ricini. Topical administration of W. somnifera seed and root extracts to last instar larvae of P. ricini disrupted moulting and metamorphosis, leading to a number of developmental abnormalities such as delay in larval-pupal and pupal-adult ecdysis, formation of larval-pupal, pupal-adult and larval-pupal-adult mosaics/chimeras, ecdysial failure, suppression of pupation and adult emergence and formation of abnormal pupae and adultoids. The treatment with seed extracts was more severe than that of root extracts as it completely suppressed the pupation and adult emergence. The results clearly suggest that the medicinal plant, W. somnifera acts as a potential insect growth regulatory (IGR) disrupting the moulting and metamorphosis as a consequence of interference with the endocrine system.

Bioefficacy of root extracts of a medicinal plant, Withania somnifera (Dunal) against a polyphagous pest, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Oral administration of root extracts of a medicinal plant, Ashwagandha (Withania somnifera) to last instar larvae of a polyphagous pest, Spodoptera litura resulted into abnormal morphogenesis and the effects comprised mortality, delay in larval-pupal and pupal-adult ecdysis, ecdysial stasis, formation of larval-pupal and pupal-adult intermediates, reduced pupation and formation of abnormal pupae, complete suppression of normal adult emergence and formation of adultoids. These effects are similar to those produced by the administration of JHAs and may be due to interference with the normal hormonal mechanism of moulting and metamorphosis.

Two Leptinotarsa uridine diphosphate N-acetylglucosamine pyrophosphorylases are specialized for chitin synthesis in larval epidermal cuticle and midgut peritrophic matrix

Uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (UAP) is involved in the biosynthesis of chitin, an essential component of the epidermal cuticle and midgut peritrophic matrix (PM) in insects. In the present paper, two putative LdUAP genes were cloned in Leptinotarsa decemlineata. In vivo bioassay revealed that 20-hydroxyecdysone (20E) and an ecdysteroid agonist halofenozide activated the expression of the two LdUAPs, whereas a decrease in 20E by RNA interference (RNAi) of an ecdysteroidogenesis gene LdSHD and a 20E signaling gene LdFTZ-F1 repressed the expression. Juvenile hormone (JH), a JH analog pyriproxyfen and an increase in JH by RNAi of an allatostatin gene LdAS-C downregulated LdUAP1 but upregulated LdUAP2, whereas a decrease in JH by silencing of a JH biosynthesis gene LdJHAMT had converse effects. Thus, expression of LdUAPs responded to both 20E and JH. Moreover, knockdown of LdUAP1 reduced chitin contents in whole larvae and integument samples, thinned tracheal taenidia, impaired larval–larval molt, larval-pupal ecdysis and adult emergence. In contrast, silencing of LdUAP2 significantly reduced foliage consumption, decreased chitin content in midgut samples, damaged PM, and retarded larval growth. The resulting larvae had lighter fresh weights, smaller body sizes and depleted fat body. As a result, the development was arrested. Combined knockdown of LdUAP1 and LdUAP2 caused an additive negative effect. Our data suggest that LdUAP1 and LdUAP2 have specialized functions in biosynthesizing chitin in the epidermal cuticle and PM respectively in L. decemlineata.

Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori.

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori.

Effect of the juvenile hormone agonist pyriproxyfen on larval and pupal development of the citrus swallowtail Papilio demoleus (Lepidoptera: Papilionidae)

The citrus swallowtail Papilio demoleus L. is a serious pest of citrus in many regions of the world. We evaluated the effects of pyriproxyfen, a juvenile hormone agonist, on late-stage larval and pupal development of the pest. Fourth and fifth instars, as well as freshly formed prepupae were treated topically with concentrations of 7.5—60 μg/|μl of the juvenile hormone analogue. Effects included prolongation of larval duration, delay in larval-larval and larval-pupal ecdysis, mortality, formation of mosaics with larval, pupal and adult characteristics, decreased and imperfect pupation, and complete inhibition of emergence. These results demonstrate that pyriproxyfen has an adverse effect on the development and metamorphosis of the citrus swallowtail, and further work is recommended, including laboratory and field studies.

Diofenolan: a novel insect growth regulator in common citrus butterfly, Papilio demoleus

Penultimate and last instar larvae of the common citrus swallowtail butterfly, Papilio demoleus L. (Papilionidae: Lepidoptera), were treated with different doses of a novel juvenoid, diofenolan. Several deformities were observed as a result of topical administration of diofenolan which include delay in larval–larval and larval–pupal ecdysis, ecdysial failure, mortality, severe reduction in pupation, deformed pupae and complete inhibition of adult emergence. The juvenoid diofenolan severely hampers the normal growth, development and metamorphosis of P. demoleus and can be incorporated into integrated pest management programs for successful control of this important pest of citrus all over the world.

Rectal sac distention is induced by 20-hydroxyecdysone in the pupa of Bombyx mori

Holometabolous insects do not excrete but store metabolic wastes during the pupal period. The waste is called meconium and is purged after adult emergence. Although the contents of meconium are well-studied, the developmental and physiological regulation of meconium accumulation is poorly understood. In Bombyx mori, meconium is accumulated in the rectal sac; thereby, the rectal sac distends at the late pupal stage. Here, we show that rectal sac distention occurs between 4 and 5 days after pupation. The distention is halted by brain-removal just after larval–pupal ecdysis but not by brain-removal 1 day after pupation. In the pupae, brain-removal just after ecdysis kept the hemolymph ecdysteroid titer low during early and mid-pupal stages. An injection of 20-hydroxyecdysone (20E) evoked the distention that was halted by brain-removal in a dose-dependent manner. Therefore, brain-removal caused the lack of ecdysteroid, and rectal sac distention did not appear in the brain-removed pupae because of the lack of ecdysteroid. We conclude that rectal sac distention is one of the developmental events regulated by 20E during the pupal period in B. mori.

BHLH-PAS family transcription factor methoprene-tolerant plays a key role in JH action in preventing the premature development of adult structures during larval–pupal metamorphosis

The biological actions of juvenile hormones are well studied; they regulate almost all aspects of an insect’s life. However, the molecular actions of these hormones are not well understood. Recent studies in the red flour beetle, Tribolium castaneum, demonstrated the utility of this insect as a model system to study JH action. These studies confirmed that the bHLH-PAS family transcription factor, methoprene-tolerant (TcMet,) plays a key role in JH action during larval stages. In this study, we investigated the role of TcMet in JH action during larval–pupal metamorphosis. The phenotypes of TcMet RNAi insects shared similarity with the phenotypes of some allatectomized lepidopteran larvae that were attempting to undergo precocious larval–pupal metamorphosis. Knocking-down TcMet during the final instar also disrupted larval–pupal ecdysis, resulting in the development of adultoid underneath the larval skin. However, the loss of TcMet did not completely block remodeling of internal tissues such as midgut. T. castaneum larvae injected with TcMet dsRNA demonstrated a resistance to a JH analog (JHA), hydroprene, irrespective of time and route of application. Knocking-down TcMet also caused down regulation of JH-response genes, JHE and Kr-h1 suggesting that TcMet might be involved in the expression of these genes. Based on the phenotype, gene expression, and JHA action studies in TcMet RNAi insects, this study concludes that Met plays a key role in JH action for preventing the premature development of adult structures during larval–pupal metamorphosis.

Appearance of Apoptotic Cells and Granular Cells in the Silkworm Midgut Lumen During Larval-Pupal Ecdysis

To study midgut degradation and programmed cell death, we performed methyl green-pyronin staining and Giemsa staining of the midgut of silkworms during metamorphosis. Midgut epithelial cells underwent pyknosis and cytoplasmic shrinkage on the second day of spinning. In the prepupal stage, all midgut epithelial cells desquamated into the midgut lumen, rapidly forming apoptotic bodies. The number of apoptotic bodies in the midgut decreased rapidly from the prepupal stage to the third day of the pupal stage. DNA fragmentation at the time of apoptotic body formation was confirmed by the comet assay. In the midgut lumen from the prepupal stage to the first through third days of the pupal stage in which apoptotic bodies were observed, granular cells were present. Their morphology was similar to that in the body fluid and, during the pupal stage, intracellular granules increased in size and number with time, giving the appearance of a foamy cell. In this stage, numerous granular cells were observed under the basement membrane of the midgut, and phagocytosed apoptotic bodies were seen within granular cells in the midgut lumen. Granular cells may be actively involved in the clearance of apoptotic bodies from the midgut during larval-pupal ecdysis.

Casein Kinases I of the Silkworm, Bombyx mori: Their Possible Roles in Circadian Timing and Developmental Determination

Doubletime (DBT), a homolog of casein kinase Iepsilon (CKIepsilon), is an essential circadian clock component and developmental regulator in Drosophila melanogaster. The authors cloned a dbt homolog from the silkworm, Bombyx mori(Bmdbt), and examined its spatial and temporal expression in comparison to a CKI[alpha] homolog (BmCKIalpha). Four Bmdbt splice variants and 2 BmCKIalpha splice variants were detected, and their expression patterns varied in different tissues. The level of Bmdbt transcript in the brain was constant under LD 12:12 while those of BmCKIalpha transcripts fluctuated with a decrease at ZT12. In situ hybridization showed presumably identical distribution of dbt, CKIalpha, and per transcripts in the putative clock neurons of the head ganglia, as well as in the retina, where CKI-and PER-like immunoreactivities were colocalized, suggesting a possible involvement of both CKIs in the B. mori circadian system. Signals were detected at 4 Ia(1) neurons in each dorsolateral protocerebrum, 6 to 8 cells in the pars intercerebralis, about 6 cells in the suboesophageal ganglion, 2 neurons in the frontal ganglion, and most of the photoreceptors. All these cells contained dbt, CKIalpha, and per antisense transcripts. The Northern analysis of dbtand CKIalpha transcripts at different developmental stages showed that both genes were expressed at relatively high levels during early embryogenesis and in the ovary. The levels of CKIalpha transcripts were also high in the late larval stages until the mid-fifth instar and then suddenly disappeared before larval-pupal ecdysis. In contrast, the transcriptional activity of both genes was low in diapausing eggs.

NEW FINDINGS ON THE DEVELOPMENTAL PROCESS OF ASCOGREGARINA TAIWANENSIS AND ASCOGREGARINA CULICIS IN AEDES ALBOPICTUS AND AEDES AEGYPTI

Infection in different stages of larvae of Aedes aegypti and Ae. albopictus with Ascogregarina taiwanensis and A. culicis, respectively, revealed that the oocysts of Ascogregarina spp. are able to infect any instar and can complete their life cycle within 9.5 +/- 1 days. When early instars ingested oocysts, parasite development was synchronized to larval-pupal ecdysis and oocyst dissemination occurred at the time of adult emergence, oviposition, or both. The parasites also developed normally when infecting 2nd, 3rd, and early 4th instars and oocysts were released only during oviposition. The parasitic development stopped at the gamont stage when oocysts were ingested by late 4th instars (6 days old). The release of sporozoites in the midgut of any larval stage started within 45 min of oocyst ingestion. About 98% of oocysts of both A. taiwanensis and A. culicis were emptied within 2-3 h of their ingestion in their respective hosts. The oocysts of both species remained viable on desiccated filter paper stored at 27 degrees C and 65 +/- 5% relative humidity, indicating that the oocysts were resistant to dryness. The oocysts of A. culicis could survive up to 6 months, whereas those of A. taiwanensis survived up to 4 months. These biological characteristics relating to parasite development might enhance the distribution of Ascogregarina spp. widely in nature and facilitate the species to be considered for biological control of Aedes mosquitoes in the future.

Effect of the bee glue (propolis) on the calorimetrically measured metabolic rate and metamorphosis of the greater wax moth Galleria mellonella

Among the moth pests of the honeybee, the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) causes the greatest damage, unless controlled at an early stage, because it feeds on wax, pollen, and cocoon of the bee larvae. This leads to the destruction of honeycomb and subsequent deterioration of weakened colonies. For controlling the pest, natural products are second to none, not least because the use of synthetic substances carries with it the problem of residues, which remain in the beehive to affect the bee products. This paper reports the results of calorimetric investigations on the effects of the bee natural insecticidal glue, propolis, on pupal metamorphosis and the metabolic rate of different larval instars.Experiments were performed by batch calorimetry to record the heat flow rate of individual larvae/pupae before and after the treatment, which consisted of dipping L5, L6, and L7 instars in a graded series of different concentrations of ethanol-dissolved propolis for 30s before blotting them. The heat production rates were then recorded for 6–7h (short period experiment) or during the entire pupal metamorphosis (long period experiment).The fifth larval instar (L5) showed higher sensitivity to propolis treatment than L6 and L7 whereby total mortality was obtained by 4% propolis for L5 and 8–10% for the latter. The higher sensitivity of L5 can be accounted for by the very high mass-specific metabolic rate and the thinner and more fragile cuticle, typical of early larval stages, allowing the free transit of nonpolar toxic substances from the surroundings after being easily disrupted by components of propolis.The treatment of the late L7 stage with nonlethal doses of propolis shortened the duration of pupal metamorphosis significantly. An untreated larva required 6.8±0.8 days (mean±S.E.,n=5) between larval–pupal and pupal–adult ecdysis, whereas this time was shortened to 5.4±0.9 and 4.8±0.5 days after treatment with 1 and 2% propolis, respectively. Though all treated larvae went through larval–pupal ecdysis, 40 and 100% of those treated with 2 and 4% propolis, respectively, displayed abortion of pupal metamorphosis and died.These results indicate that propolis is toxic at higher concentrations and an insect growth regulator at lower ones. The use of propolis in the control of G. mellonella and its subsequent occurrence in honeybee products such as honey and wax may not cause the problem of a toxic residue, as it is the natural component in the beehive.