Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Downregulation of ultraspiracle gene expression delays pupal development in honeybees

Ecdysteroids regulate many aspects of insect physiology after binding to a heterodimer composed of the nuclear hormone receptor proteins ecdysone receptor (EcR) and ultraspiracle (Use). Several lines of evidence have suggested that the latter also plays important roles in mediating the action of juvenile hormone (JH) and, thus, integrates signaling by the two morphogenetic hormones. By using an RNAi approach, we show here that Us p participates in the mechanism that regulates the progression of pupal development in Apis mellifera, as indicated by the observed pupal developmental delay in usp knocked-down bees. Knock-down experiments also suggest that the expression of regulatory genes such as ftz transcription factor 1 (ftz-f1) and juvenile hormone esterase (jhe) depend on Usp. Vitellogenin (vg), the gene coding the main yolk protein in honeybees, does not seem to be under Usp regulation, thus suggesting that the previously observed induction of vg expression by JH during the last stages of pupal development is mediated by yet unknown transcription factor complexes. (C) 2008 Elsevier Ltd. All rights reserved.

A honeybee storage protein gene, hex 70a, expressed in developing gonads and nutritionally regulated in adult fat body

In preparing for metamorphosis, insect larvae store a huge amount of proteins in hemolymph, mainly hexamerins. Out of the four hexamerins present in the honeybee larvae, one, HEX 70a, exhibited a distinct developmental pattern, especially since it is also present in adults. Here, we report sequence data and experimental evidence suggesting alternative functions for HEX 70a, besides its well-known role as an amino acid resource during metamorphosis. The hex 70a gene consists of 6 exons and encodes a 684 amino acid chain containing the conserved hemocyanin N, M, and C domains. HEX 70a classifies as an arylphorin since it contains more than 15% of aromatic amino acids. In the fat body of adult workers, hex 70a expression turned out to be a nutrient-limited process. However, the fat body is not the only site for hex 70a expression. Both, transcript and protein subunits were also detected in developing gonads from workers, queens and drones, suggesting a role in ovary differentiation and testes maturation and functioning. In its putative reproductive role, HEX 70a however differs from the yolk protein, vitellogenin, since it was not detected in eggs or embryos. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

Co-occurrence of three types of egg policing in the Norwegian wasp Dolichovespula norwegica

In insect societies, workers often try to challenge the reproductive monopoly of the queen by laying their own eggs. Successful worker reproduction, however, is frequently prevented by queen policing or worker policing, whereby either the mother queen or non-reproductive workers selectively kill worker-laid eggs. Recently, a third mechanism-""selfish"" worker policing-has also been described in which the workers selectively police worker-laid eggs but also lay eggs themselves. Here, we present results from the monogynous wasp Dolichovespula norwegica, which show that all three kinds of policing-queen policing, worker policing and ""selfish"" worker policing-co-occur. The net effect of these three kinds of policing collectively favoured the queen`s reproduction, as within 1 day 44% of the worker-laid eggs versus only 8% of the queen-laid eggs were eaten. Of the worker-laid eggs that were killed by workers, approximately two thirds were eaten by the reproductive workers even though these made up only a small proportion, 8%, of the work force. This means that policing workers obtained both direct fitness benefits as well as indirect (inclusive) fitness. In addition, we show that worker policing was carried out by a limited, specialised set of workers that was estimated to constitute approximately one quarter of the whole colony and of which 66% were non-reproductive.

The juvenile hormone (JH) epoxide hydrolase gene in the honey bee (Apis mellifera) genome encodes a protein which has negligible participation in JH degradation

Epoxide hydrolases are multifunctional enzymes that are best known in insects for their role in juvenile hormone (JH) degradation. Enzymes involved in JH catabolism can play major roles during metamorphosis and reproduction, such as the JH epoxide hydrolase (JHEH), which degrades JH through hydration of the epoxide moiety to form JH diol, and JH esterase (JHE), which hydrolyzes the methyl ester to produce JH acid. In the honey bee, JH has been co-opted for additional functions, mainly in caste differentiation and in age-related behavioral development of workers, where the activity of both enzymes could be important for JH titer regulation. Similarity searches for jheh candidate genes in the honey bee genome revealed a single Amjheh gene. Sequence analysis, quantification of Amjheh transcript levels and Western blot assays using an AmJHEH-specific antibody generated during this study revealed that the AmJHEH found in the fat body shares features with the microsomal JHEHs from several insect species. Using a partition assay we demonstrated that AmJHEH has a negligible role in JH degradation, which, in the honey bee, is thus performed primarily by JHE. High AmJHEH levels in larvae and adults were related to the ingestion of high loads of lipids, suggesting that AmJHEH has a role in dietary lipid catabolism. (C) 2010 Elsevier Ltd. All rights reserved.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae)

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORE of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pl of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Organization, evolution and transcriptional profile of hexamerin genes of the parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae)

Hexamerins and prophenoloxidases (PPOs) proteins are members of the arthropod-haemocyanin superfamily. In contrast to haemocyanin and PPO, hexamerins do not bind oxygen, but mainly play a role as storage proteins that supply amino acids for insect metamorphosis. We identified seven genes encoding hexamerins, three encoding PPOs, and one hexamerin pseudogene in the genome of the parasitoid wasp Nasonia vitripennis. A phylogenetic analysis of hexamerins and PPOs from this wasp and related proteins from other insect orders suggests an essentially order-specific radiation of hexamerins. Temporal and spatial transcriptional profiles of N. vitripennis hexamerins suggest that they have physiological functions other than metamorphosis, which are arguably coupled with its lifestyle.

Functional and Evolutionary Insights from the Genomes of Three Parasitoid Nasonia Species

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

TRADE-OFF BETWEEN IMMUNE STIMULATION AND EXPRESSION OF STORAGE PROTEIN GENES

Proteins stored in insect hemolymph may serve (is a source of amino acids and energy for metabolism, and development. The expression of the main storage proteins was assessed in bacterial-challenged honey bees using real-time (RT)-PCH and Western blot.. After ensuring that. the immune system had, been activated by measuring the ensuing expression (, the innate immune response genes, defensin-1 (def-1) and prophenoloxidase (pro PO), we verified the expression of four genes encoding storage proteins. The levels of vitellogenin (vg) mRNA and of the respective protein. were significantly lowered in bees injected with bacteria or water only (injury). An equivalent response was observed in orally-infected bees. The levels of apolipophorin II/I (apoLP-II/I) and hexamerin (hex 70a) mRNAs did not significantly change, but levels of Hex 70a protein subunit showed a substantial decay after bacterial challenge or injury. Infection also caused a strong reduction in the levels of apoLP-III transcripts. Our findings are consistent with a down-regulation, of the express and accumulation of storage proteins as a consequence of activation of the immune system, suggesting that this phenomenon. represents a strategy to redirect resources to combat injury or infection. (C) 2009 Wiley Periodicals, Inc.

A non-invasive method for silencing gene transcription in honeybees maintained under natural conditions

in the Apis mellifera post-genomic era, RNAi protocols have been used in functional approaches. However, sample manipulation and invasive methods such as injection of double-stranded RNA (dsRNA) can compromise physiology and survival. To circumvent these problems, we developed a non-invasive method for honeybee gene knockdown, using a well-established vitellogenin RNAi system as a model. Second instar larvae received dsRNA for vitellogenin (dsVg-RNA) in their natural diet. For exogenous control, larvae received dsRNA for GFP (dsGFP-RNA). Untreated larvae formed another control group. Around 60% of the treated larvae naturally developed until adult emergence when 0.5 mu g of dsVg-RNA or dsGFP-RNA was offered while no larvae that received 3.0 mu g of dsRNA reached pupal stages. Diet dilution did not affect the removal rates. Viability depends not only on the delivered doses but also on the internal conditions of colonies. The weight of treated and untreated groups showed no statistical differences. This showed that RNAi ingestion did not elicit drastic collateral effects. Approximately 90% of vitellogenin transcripts from 7-day-old workers were silenced compared to controls. A large number of samples are handled in a relatively short time and smaller quantities of RNAi molecules are used compared to invasive methods. These advantages culminate in a versatile and a cost-effective approach. (c) 2008 Elsevier Ltd. All rights reserved.

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.

The gall-inducing habit has evolved multiple times among the eriococcid scale insects (Sternorrhyncha : Coccoidea : Eriococcidae)

The habit of inducing plant galls has evolved multiple times among insects but most species diversity occurs in only a few groups, such as gall midges and gall wasps. This phylogenetic clustering may reflect adaptive radiations in insect groups in which the trait has evolved. Alternatively, multiple independent origins of galling may suggest a selective advantage to the habit. We use DNA sequence data to examine the origins of galling among the most speciose group of gall-inducing scale insects, the eriococcids. We determine that the galling habit has evolved multiple times, including four times in Australian taxa, suggesting that there has been a selective advantage to galling in Australia. Additionally, although most gall-inducing eriococcid species occur on Myrtaceae, we found that lineages feeding on Myrtaceae are no more likely to have evolved the galling habit than those feeding on other plant groups. However, most gall-inducing species-richness is clustered in only two clades (Apiomorpha and Lachnodius + Opisthoscelis), all of which occur exclusively on Eucalyptus s.s. The Eriococcidae and the large genus Eriococcus were determined to be non-monophyletic and each will require revision. (C) 2004 The Linnean Society of London.

The nymph of Tortopus harrisi Traver (Ephemeroptera: Polymitarcyidae)

Polymitarcyidae is a family of burrowing mayflies (Ephemeroptera: Ephemeroidea) distributed throughout the world but with highest diversity in the Neotropics. Tortopus Needham & Murphy, with a Panamerican distribution, is known from twelve species described in the adult stage. Nymphs are only known for three species: T. puella (Pictet), T. obscuripennis Dominguez and T. sarae Dominguez, and present a rather homogeneous morphology (Molineri 2008). They were firstly described for T. puella by Scott et al. (1959) and later Molineri (2008) described the other two. Both studies reported that these species burrow U-shaped tunnels in clay banks of rivers and streams, thus preventing them from being sampled in most limnological studies (that use surbers, drags, or drift nets). The aim of the present contribution is to describe and illustrate the previously unknown nymph of Tortopus harrisi Traver that shows important anatomical differences with the other nymphs known in the genus. This morphological differentiation suggests a different habitat use by these nymphs, sampled with drag and surber samplers in sandy substrate. New locality records are given for T. harrisi in Brazil. The nymphs are preserved in alcohol, mouthparts, legs and genital rudiments were mounted in microscope slides with Canada Balsam. Drawings were made with a camera lucida attached to a stereo microscope. The material is deposited in CUIC (Cornell University Insect Collection, Ithaca, NY), IML (Instituto Miguel Lillo, Tucuman) and in MZSP (Museu de Zoologia da Universidade de Sao Paulo, Sao Paulo). Catalogs and bibliography were consulted at Ephemeroptera Galactica (Hubbard 2009).

Effects of pasture implantation on the termite (Isoptera) fauna in the Central Brazilian Savanna (Cerrado)

The advance of agricultural frontier may cause the Cerrado (Brazilian savanna) to disappear before 2030. This work focuses on measuring the impact of pasture implantation on a cerrado`s termite fauna. Termites were sampled in a cerrado sensu stricto and a pasture, originally cerrado. All species were classified as their feeder group, accumulation curves were made and Shannon-Wiener indexes and beta diversity were calculated for both areas. Cerrado was richer than pasture and species composition differed considerably, leading beta diversity to a high value. The humivorous was the most representative species, followed by grass/litter feeders, xylophagous and, less representative, the intermediates. There were more xylophagous and intermediates species on cerrado than in pasture; the grass/litter feeders were more abundant in pasture, but didn`t differed in number or species; and humivorous didn`t differed neither in richness nor in abundance. This work shows that the simplification of the habitat is indeed causing the extinction of populations that depend on some specifics resource.