Effects of juvenile hormone and ecdysone on the timing of vitellogenin appearance in hemolymph of queen and worker pupae of Apis mellifera

The caste-specific regulation of vitellogenin synthesis in the honeybee represents a problem with many yet unresolved details. We carried out experiments to determine when levels of vitellogenin are first detected in hemolymph of female castes of Apis mellifera, and whether juvenile hormone and ecdysteroids modulate this process. Vitellogenin levels were measured in hemolymph using immunological techniques. We show that in both castes the appearance of vitellogenin in the hemolymph occurs during the pupal period, but the timing was different in the queen and worker. Vitellogenin appears in queens during an early phase of cuticle pigmentation approximately 60h before eclosion, while in workers the appearance of vitellogenin is more delayed, initiating in the pharate adult stage, approximately 10h before eclosion. The timing of vitellogenin appearance in both castes coincides with a slight increase in endogenous levels of juvenile hormone that occurs at the end of pupal development. The correlation between these events was corroborated by topical application of juvenile hormone. Exogenous juvenile hormone advanced the timing of vitellogenin appearance in both castes, but caste-specific differences in timing were maintained. Injection of actinomycin D prevented the response to juvenile hormone. In contrast, queen and worker pupae that were treated with ecdysone showed a delay in the appearance of vitellogenin. These data suggest that queens and workers share a common control mechanism for the timing of vitellogenin synthesis, involving an increase in juvenile hormone titers in the presence of low levels of ecdysteroids

Does trade help to explain tourism demand? The case of Portugal

The tourism industry has expanded in recent years due to internal and external environmental forces. These forces, income, trade, consumer price, and geographical distance are interconnected. Accounting for imperfect competition and increasing returns to scale, the new trade theory offers the explanation of dynamic gains from international trade. A large number of studies attempt to test the hypothesis that there is a link between trade and tourism. Most of the studies show that trade and tourism are positively correlated. This paper specifies static and dynamic panel demand models for tourism in Portugal and estimates demand equations using tourist inflow data for the period 1995-2006. We find that bilateral trade, immigration, border, and geographical distance between Portugal and countries of origin are the main determinants of tourism to Portugal. The dynamic panel data approach indicates that trade, population, and income are more important determinants than relative price

Ultraspiracle and caste differentiation in Apis mellifera

O hormônio juvenil (HJ) e os ecdisteróides, especialmente 20-hidroxiecdisona (20E), controlam muitos aspectos no desenvolvimento de insetos. Em insetos eusociais, estes hormônios estão diretamente envolvidos na diferenciação de castas alternativas. O HJ, ademais, modula o desenvolvimento e a evolução do comportamento de operárias adultas em Apis mellifera. Nesse trabalho, clonamos e sequenciamos o cDNA que codifica para Ultraspiracle de Apis mellifera (AmUsp) e analisamos a resposta de usp a ambos hormônios morfogenéticos. Amusp parece ser um gene de cópia simples que produz dois transcritos (~4 e 5kb) que são diferencialmente expressos no corpo do animal. AmUsp mostra maior similaridade respeito aos ortólogos de Locusta, Celuca e vertebrados do que aos do resto de insetos. Estas características e sua rápida resposta transcricional a HJ sugere que algumas funções do Usp de Apis mellifera podem depender de ligande. Por outro lado, usando RNAi testamos a resposta de vários genes à diminuição da expressão de usp durante o desenvolvimento pupal de operárias. Enquanto a expressão de vg não foi claramente alterada em pupas knock-down para usp (KDusp), os genes para a pro-fenoloxidase e para a esterase do HJ foram inibidos. De maneira interessante, a expressão de ftz-f1 (um receptor nuclear órfão) também foi diminuída em pupas KDusp, sugerindo a participação de Usp na regulação da expressão destes genes. Títulos baixos de HJ durante os últimos estágios do desenvolvimento larval foram sugeridos como os responsáveis por desencadear a morte celular programada em ovários de operárias, ao ponto tal que só uns poucos ovaríolos permanecem para formar parte do sistema reprodutivo destas fêmeas. Nós determinamos o perfil de expressão de usp nos ovários de rainhas e operárias durante os últimos estágios do desenvolvimento larval. Análises por RT-PCR semiquantitativa mostraram que o mRNA de usp segue de perto os títulos hemolinfáticos de HJ, e que os maiores níveis de expressão deste gene encontrados em L4 de rainhas se correspondem com títulos maiores deste hormônio. Porém, experimentos de imunofluorescência utilizando um anticorpo monoclonal contra Usp de Drosophila melanogaster, mostraram estruturas marcadas somente na região de formação de clasters nos ovaríolos de operárias se preparando para a metamorfose. Esta região corresponde ao centro de células germinativas onde ocorrem os mais evidentes eventos de morte celular. Este padrão de expressão de usp sugere sua participação nestes processos, sozinho, ou formando heterodímeros com outros fatores de transcrição. Estes dados e a resposta positiva de usp a altos títulos de HJ nos permitem começar a elaboração de um modelo conceitual da rede de expressão gênica promovida pelo HJ durante o desenvolvimento e diferenciação de castas de Apis mellifera.In addition to their general roles in insect development and reproduction, juvenile hormone (JH) and ecdysone (20E) coordinate specific processes in social insects. They are directly involved in the differentiation of the alternative caste phenotypes, and, particularly in the honey bee, Apis mellifera, they modulate behavioral development in adult workers. In the present study, we have cloned and sequenced a cDNA encoding Apis mellifera Ultraspiracle (AmUsp), and analyzed its responses to both morphogenetic hormones, JH and 20E. Amt/sp seems to be a single copy gene that produces two transcripts (~4 and 5kb) that are differentially expressed in the different tissues and organs of a bee. In its amino acid sequence, AmUsp shows greater similarity to its orthologs from the vertebrate-crab-locust group than to the dipteran-lepidopteran group. These characteristics and its rapid up-regulation by JH suggest that some of the Apis mellifera Usp functions may depend on ligand binding. To investigate possible target genes regulated by AmUsp, we have used an iRNA technique to down-regulate usp expression during pupal development of Apis mellifera workers. This allowed us to test the response of ftz-f1, juvenile hormone esterase (jhe), pro-phenoloxidase (proPo) and vitellogenin (vg) genes to decreased levels of usp expression. While vg expression levels were not clearly altered in usp knock-down pupae (kd), the proPo and the genes were down regulated. Interestingly, ftz-f1 (an orphan nuclear receptor) also turned out to be down regulated in kd pupae, indicating Usp protein participation in controlling the expression of these genes. A low JH titer during the final stages of larval development has been shown to trigger programmed cell death in the ovaries of worker larvae to the point that only a few ovarioles remain to form the functional reproductive system of these females. Hence, we determined the AmUsp expression pattern in the ovaries of queen and worker honeybees during the late stages of larval development. RT-PCR analyses showed that usp expression closely follows the haemolymph JH titer, and that the higher levels of usp expression in L4 queens reflect the higher titers of this hormone. Immunocytochemistry experiments with a mAb raised against a dipteran Usp protein revealed stained structures only in the region of cluster formation in ovarioles of worker larvae preparing for metamorphosis. This region corresponds to the germ cell center where processes of cell death and proliferation have been shown to occur. The pattern of usp expression suggests its participation in these processes, either alone or by heterodimerization with other transcription factors. These reports and the known positive response of usp to high levels of JH allow us to start building a conceptual model for the gene expression network promoted by JH during honey bee development and caste differentiation

Host preference of the bean weevil Zabrotes subfasciatus

It is largely known that the range of an insect diet is mostly determined by oviposition behavior, mainly in species with endophytic larvae such as Zabrotes subfasciatus. However, the proximate factors determining host choice and the subsequent steps leading to the expansion or reduction of the host number and occasional host shifts are largely unknown. We analyzed various factors determining host preference of Z. subfasciatus through the evaluation of: (i) oviposition preference of a wild population of Z subfasciatus on the usual host (bean) and unusual hosts (lentil, chickpea and soy), and the performance of the offspring; (ii) artificial selection for increasing preference for hosts initially less frequently chosen; (iii) comparison of oviposition behavior between two different populations (reared for similar to 30 generations in beans or chickpeas, respectively); (iv) oviposition timing on usual and unusual hosts; and (v) identification of preference hierarchies. We found that when using unusual hosts, there is no correlation between performance and preference and that the preference hierarchy changes only slightly when the population passes through several generations on the less frequently accepted host. We also found a positive response to artificial selection for increasing oviposition on the less preferred host; however, when the host-choice experiment involved two varieties of the usual host, the response was faster than when the choice involved usual and unusual hosts. Finally, beetles reared on an unusual host (chickpea) for 26 generations showed similar good fitness on both usual and unusual hosts, indicating that the use of a new host does not necessarily result in the loss of performance on the original host. Nevertheless, this population showed lower fitness on the usual host than that of the original population, suggesting an underlying partial trade-off phenomenon which may contribute to a broadening of diet of this insect species

Ultraspiracle of the stingless bees Melipona scutellaris and Scaptotrigona depilis: cDNA sequence and expression profiles during pupal development

Reproduction and development in insects and caste differentiation in bees are mainly governed by ecdysteroids and juvenile hormone. We characterized the ultraspiracle (nuclear hormone receptor) cDNA of the highly social bees Melipona scutellaris and Scaptotrigona depilis. The predicted Usp proteins (with 427aa) show a greater sequence similarity to its orthologs from the “ApLoTe” group than to the dipteran-lepidopteran group, suggesting that Usp proteins included in the bee group might share functional characteristics. The expression profiles in fat bodies (FB) and brains turned out not to be very similar between the species. In M. scutellaris FB and brain and in S. depilis brain usp expression anti-parallels pupal ecdysteroid titers, thus suggesting a repression of usp expression by this hormone

Characteristics of stages of female honeybee larval development.

<p>W: workers;</p><p>Q: queens.</p

Immunolocalization of Ubx (FP6.87 antibody) in honeybee white-eyed pupale hind legs.

<p><b>A-C:</b> Ubx is expressed in the tibia and basitarsi of workers. There is a region in the tibia (which may be the future corbicula) that does not express Ubx. <b>D-I:</b> In the basitarsus and distal portion of the tibia (arrowhead) in workers, there are double nuclei that do not express Ubx, arranged in a similar pattern as that of the bristles in the adult hind leg. <b>J-L:</b> In the hind legs of queen white-eyed pupae, Ubx is expressed only in the basitarsi. In blue: DAPI; in red: Ubx; Btar: basitarsi; Tb: tibia. Original scale bars of confocal system.</p