A comparative analysis reveals weak relationships between ecological factors and beta diversity of stream insect metacommunities at two spatial levels

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Subacute toxicity assessment of diflubenzuron, an insect growth regulator, in adult male rats

Difubenzuron (DFB), an insecticide and acaricide insect growth regulator, can be used in agriculture against insect predators and in public health programs, to control insects and vectors, mainly Aedes aegypti larvae. Due to the lack of toxicological assessments of this compound, the objective of the present study was to evaluate the toxicological effects of subacute exposure to the DFB insecticide in adult male rats. Adult male rats were exposed (gavage) to 0, 2, 4, or 8 mg/kg of DFB for 28 days. No clinical signs of toxicity were observed in the DFB-treated animals of the experimental groups. However, there was an increase in serum levels of alanine aminotransferase in the group that received 8 mg/Kg/DFB/day and urea at doses of 4 and 8 mg/Kg/DFB/day, without altering other biochemical or hematological parameters. The subacute exposure to the lowest dose of DFB caused significant decrease in testis weight, daily sperm production, and in number of sperm in the epididymis in relation to the control group. However, no alterations were observed in the sperm morphology, testicular, epididymis, liver and kindney histology, or testosterone levels. These findings unveiled the hazardous effects of DFB on male reproduction after the subacute exposure and special attention should be addressed to the effects of low doses of this pesticide.

Contributions of demography and dispersal parameters to the spatial spread of a stage-structured insect invasion

Stage-structured models that integrate demography and dispersal can be used to identify points in the life cycle with large effects on rates of population spatial spread, information that is vital in the development of containment strategies for invasive species. Current challenges in the application of these tools include: (1) accounting for large uncertainty in model parameters, which may violate assumptions of ‘‘local’’ perturbation metrics such as sensitivities and elasticities, and (2) forecasting not only asymptotic rates of spatial spread, as is usually done, but also transient spatial dynamics in the early stages of invasion. We developed an invasion model for the Diaprepes root weevil (DRW; Diaprepes abbreviatus [Coleoptera: Curculionidae]), a generalist herbivore that has invaded citrus-growing regions of the United States. We synthesized data on DRW demography and dispersal and generated predictions for asymptotic and transient peak invasion speeds, accounting for parameter uncertainty. We quantified the contributions of each parameter toward invasion speed using a ‘‘global’’ perturbation analysis, and we contrasted parameter contributions during the transient and asymptotic phases. We found that the asymptotic invasion speed was 0.02–0.028 km/week, although the transient peak invasion speed (0.03– 0.045 km/week) was significantly greater. Both asymptotic and transient invasions speeds were most responsive to weevil dispersal distances. However, demographic parameters that had large effects on asymptotic speed (e.g., survival of early-instar larvae) had little effect on transient speed. Comparison of the global analysis with lower-level elasticities indicated that local perturbation analysis would have generated unreliable predictions for the responsiveness of invasion speed to underlying parameters. Observed range expansion in southern Florida (1992–2006) was significantly lower than the invasion speed predicted by the model. Possible causes of this mismatch include overestimation of dispersal distances, demographic rates, and spatiotemporal variation in parameter values. This study demonstrates that, when parameter uncertainty is large, as is often the case, global perturbation analyses are needed to identify which points in the life cycle should be targets of management. Our results also suggest that effective strategies for reducing spread during the asymptotic phase may have little effect during the transient phase. Includes Appendix.

Plastic pellets as oviposition site and means of dispersal for the ocean-skater insect Halobates

Microplastics are omnipresent in the oceans and generally have negative impacts on the biota. However, flotsam may increase the availability of hard substrates, which are considered a limiting resource for some oceanic species, e.g. as oviposition sites for the ocean insect Halobates. This study describes the use of plastic pellets as an oviposition site for Halobates micans and discusses possible effects on its abundance and dispersion. Inspection of egg masses on stranded particles on beaches revealed that a mean of 24% (from 0% to 62%) of the pellets bore eggs (mean of 5 and max. of 48 eggs per pellet). Most eggs (63%) contained embryos, while 37% were empty egg shells. This shows that even small plastic particles are used as oviposition site by H. micans, and that marine litter may have a positive effect over the abundance and dispersion of this species. (C) 2012 Elsevier Ltd. All rights reserved.

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Mutualism from the inside: coordinated development of plant and insect in an active pollinating fig wasp

Recent studies on the obligate interaction between fig trees and their pollinating agaonid wasps have focused on population aspects and wasp-seed exploitation at the level of the inflorescence. Detailed studies on larval and gall development are required to more fully understand how resources are exploited and adaptations fine-tuned by each partner in nursery pollination mutualisms. We studied the larval development of the active pollinating fig wasp, Pegoscapus sp., and the galling process of individual flowers within the figs of its monoecious host, Ficus citrifolia, in Brazil. The pollinator development is strongly dependent on flower pollination. Figs entered by pollen-free wasps were in general more likely to abort. Retained, unpollinated figs had both higher larval mortality and a lower number of wasps. Pegoscapus sp. larvae are adapted to plant development, with two contrasting larval feeding strategies proceeding alongside gall development. The first two larval stages behave as ovary parasites. Later larval stages feed on hypertrophied endosperm. This indicates that a successful galling process relies on endosperm, and also reveals why pollination would be a prerequisite for the production of high-quality galls for this Pegoscapus species.

Sugarwin: A Sugarcane Insect-Induced Gene with Antipathogenic Activity

In sugarcane fields, colonization of the stalk by opportunistic fungi usually occurs after the caterpillar Diatraea saccharalis attacks the sugarcane plant. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN), sugarcane wound-inducible proteins SUGARWIN1 and SUGARWIN2, have been identified in sugarcane by an in silico analysis. Antifungal properties have been described for a number of BARWIN homologues. We report that a SUGARWIN:green fluorescent protein fusion protein is located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of sugarwin transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment. The accumulation of transcripts is late induced and is restricted to the site of the wound. Although the transcripts of sugarwin genes were strongly increased following insect attack, the protein itself did not show any effect on insect development; rather, it altered fungal morphology, leading to the apoptosis of the germlings. These results suggest that, in the course of evolution, sugarwin-encoding genes were recruited by sugarcane due to their antipathogenic activity. We rationalize that sugarcane is able to induce sugarwin gene expression in response to D. saccharalis feeding as a concerted plant response to the anticipated invasion by the fungi that typically penetrate the plant stalk after insect damage.

Comparing assemblages of Asteraceae and their insect herbivores under different land-use regimens

Accelerated tropical landscape changes occurring over recent decades have produced environmental mosaics comprising remaining isolated green areas and mixed land-use types. Our objective was to study the effects of alterations in the natural landscape on the species composition and structure of assemblages of Asteraceae and their endophagous insects through comparisons between cerrado (savanna), pastures and Eucalyptus stands. We first investigated whether similarities between assemblages of Asteraceae and their insects varied among land uses or localities. Secondly, we asked whether assemblages of Eucalyptus stands and pastures are subsets of those within the cerrado. We sampled within randomly deployed transects in 15 areas. Land use was found to be an important factor in determining plant composition similarity; however, locality did not exert any significant influence. Pastures were less similar to one another, suggesting high beta diversity. Similarities among insect assemblages were correlated with plant assemblage composition, but not with land use or locality. Species of Tephritidae were distributed along localities independently of land use. High beta diversity in Asteraceae assemblages among cerrados and pastures was supported by nestedness analysis. Plant assemblages in Eucalyptus stands were subsets of cerrado, but pasture assemblages were only partial subsets. A higher degree of nestedness in insect assemblages than in plant assemblages indicated lower beta diversity within these herbivores. Our data indicate that many herbivores are specialized on widely distributed plant genera. Conservation of Asteraceae species and their flower head insects depends not only on maintenance of landscape fragments but also on the correct matching of management form and land use. Such management may contribute to reducing isolation of plant and insect species by increasing the connectivity of remaining cerrado tracts, allowing population maintenance even at low abundances.

Analysis of Insect Cuticular Compounds by Non-lethal Solid Phase Micro Extraction with Styrene-Divinylbenzene Copolymers

Insect cuticular hydrocarbons including relatively non-volatile chemicals play important roles in cuticle protection and chemical communication. The conventional procedures for extracting cuticular compounds from insects require toxic solvents, or non-destructive techniques that do not allow storage of subsequent samples, such as the use of SPME fibers. In this study, we describe and tested a non-lethal process for extracting cuticular hydrocarbons with styrene-divinylbenzene copolymers, and illustrate the method with two species of bees and one species of beetle. The results demonstrate that these compounds can be efficiently trapped by ChromosorbA (R) (SUPELCO) and that this method can be used as an alternative to existing methods.

Head-to-Head Comparison of Three Vaccination Strategies Based on DNA and Raw Insect-Derived Recombinant Proteins against Leishmania

Parasitic diseases plague billions of people among the poorest, killing millions annually, and causing additional millions of disability-adjusted life years lost. Leishmaniases affect more than 12 million people, with over 350 million people at risk. There is an urgent need for efficacious and cheap vaccines and treatments against visceral leishmaniasis (VL), its most severe form. Several vaccination strategies have been proposed but to date no head-to-head comparison was undertaken to assess which is the best in a clinical model of the disease. We simultaneously assayed three vaccination strategies against VL in the hamster model, using KMPII, TRYP, LACK, and PAPLE22 vaccine candidate antigens. Four groups of hamsters were immunized using the following approaches: 1) raw extracts of baculovirus-infected Trichoplusia ni larvae expressing individually one of the four recombinant proteins (PROT); 2) naked pVAX1 plasmids carrying the four genes individually (DNA); 3) a heterologous prime-boost (HPB) strategy involving DNA followed by PROT (DNA-PROT); and 4) a Control including empty pVAX1 plasmid followed by raw extract of wild-type baculovirus-infected T. ni larvae. Hamsters were challenged with L. infantum promastigotes and maintained for 20 weeks. While PROT vaccine was not protective, DNA vaccination achieved protection in spleen. Only DNA-PROT vaccination induced significant NO production by macrophages, accompanied by a significant parasitological protection in spleen and blood. Thus, the DNA-PROT strategy elicits strong immune responses and high parasitological protection in the clinical model of VL, better than its corresponding naked DNA or protein versions. Furthermore, we show that naked DNA coupled with raw recombinant proteins produced in insect larvae biofactories -the cheapest way of producing DNA-PROT vaccines-is a practical and cost-effective way for potential "off the shelf" supplying vaccines at very low prices for the protection against leishmaniases, and possibly against other parasitic diseases affecting the poorest of the poor.

Genes involved in thoracic exoskeleton formation during the pupal-to-adult molt in a social insect model, Apis mellifera

Background: The insect exoskeleton provides shape, waterproofing, and locomotion via attached somatic muscles. The exoskeleton is renewed during molting, a process regulated by ecdysteroid hormones. The holometabolous pupa transforms into an adult during the imaginal molt, when the epidermis synthe3sizes the definitive exoskeleton that then differentiates progressively. An important issue in insect development concerns how the exoskeletal regions are constructed to provide their morphological, physiological and mechanical functions. We used whole-genome oligonucleotide microarrays to screen for genes involved in exoskeletal formation in the honeybee thoracic dorsum. Our analysis included three sampling times during the pupal-to-adult molt, i.e., before, during and after the ecdysteroid-induced apolysis that triggers synthesis of the adult exoskeleton. Results: Gene ontology annotation based on orthologous relationships with Drosophila melanogaster genes placed the honeybee differentially expressed genes (DEGs) into distinct categories of Biological Process and Molecular Function, depending on developmental time, revealing the functional elements required for adult exoskeleton formation. Of the 1,253 unique DEGs, 547 were upregulated in the thoracic dorsum after apolysis, suggesting induction by the ecdysteroid pulse. The upregulated gene set included 20 of the 47 cuticular protein (CP) genes that were previously identified in the honeybee genome, and three novel putative CP genes that do not belong to a known CP family. In situ hybridization showed that two of the novel genes were abundantly expressed in the epidermis during adult exoskeleton formation, strongly implicating them as genuine CP genes. Conserved sequence motifs identified the CP genes as members of the CPR, Tweedle, Apidermin, CPF, CPLCP1 and Analogous-to-Peritrophins families. Furthermore, 28 of the 36 muscle-related DEGs were upregulated during the de novo formation of striated fibers attached to the exoskeleton. A search for cis-regulatory motifs in the 5′-untranslated region of the DEGs revealed potential binding sites for known transcription factors. Construction of a regulatory network showed that various upregulated CP- and muscle-related genes (15 and 21 genes, respectively) share common elements, suggesting co-regulation during thoracic exoskeleton formation. Conclusions: These findings help reveal molecular aspects of rigid thoracic exoskeleton formation during the ecdysteroid-coordinated pupal-to-adult molt in the honeybee.

Molecular and biological analysis on the horizontal transfer of insect transposons in Baculoviruses

Summary During the infection of Lepidoptera larvae with baculoviruses the horizontal escape of Tc1-like transposons, termed TCl4.7 and TCp3.2, from the genome of the host Cryptophlebia leucotreta and Cydia pomonella into the genome of Cydia pomonella granulovirus was observed. In this study we addressed the question whether the transposon harboring viruses had a replication advantage over the wild-type and became dominant in the virus population or whether the activity of the host transposable elements is stimulated by virus infection. Biological characterization studies demonstrated that the transposon containing viruses killed C. pomonella larvae slower than CpGV-M. In co-infection experiments of C. pomonella larvae using a mixture of CpGV-M and mutant viruses as inoculum, it was shown that the transposon carrying mutants had a significant selection disadvantage compared to CpGV-M. Transcription levels of the transposase gene of TCp3.2 were investigated in virus infected and uninfected larvae. These experiments demonstrated that a higher level of transposase transcription was detectable in CpGV-M infected than in mock infected control larvae. This observation gave strong evidence that CpGV-M infection might trigger the activity of transposon TCp3.2 within the genome of Cydia pomonella. Our results suggest that the horizontal transfer of insect host transposons into baculovirus genomes might be induced by virus infection.

Comparative phylogeography of two co-distributed arctic-alpine freshwater insect species in Europe

The distribution pattern of European arctic-alpine disjunct species is of growing interest among biogeographers due to the arising variety of inferred demographic histories. In this thesis I used the co-distributed mayfly Ameletus inopinatus and the stonefly Arcynopteryx compacta as model species to investigate the European Pleistocene and Holocene history of stream-inhabiting arctic-alpine aquatic insects. I used last glacial maximum (LGM) species distribution models (SDM) to derive hypotheses on the glacial survival during the LGM and the recolonization of Fennoscandia: 1) both species potentially survived glacial cycles in periglacial, extra Mediterranean refugia, and 2) postglacial recolonization of Fennoscandia originated from these refugia. I tested these hypotheses using mitochondrial sequence (mtCOI) and species specific microsatellite data. Additionally, I used future SDM to predict the impact of climate change induced range shifts and habitat loss on the overall genetic diversity of the endangered mayfly A. inopinatus.rnI observed old lineages, deep splits, and almost complete lineage sorting of mtCOI sequences between mountain ranges. These results support the hypothesis that both species persisted in multiple periglacial extra-Mediterranean refugia in Central Europe during the LGM. However, the recolonization of Fennoscandia was very different between the two study species. For the mayfly A. inopinatus I found strong differentiation between the Fennoscandian and all other populations in sequence and microsatellite data, indicating that Fennoscandia was recolonized from an extra European refugium. High mtCOI genetic structure within Fennoscandia supports a recolonization of multiple lineages from independent refugia. However, this structure was not apparent in the microsatellite data, consistent with secondary contact without sexual incompability. In contrast, the stonefly A. compacta exhibited low genetic structure and shared mtCOI haplotypes among Fennoscandia and the Black Forest, suggesting a shared Pleistocene refugium in the periglacial tundrabelt. Again, there is incongruence with the microsatellite data, which could be explained with ancestral polymorphism or female-biased dispersal. Future SDM projects major regional habitat loss for the mayfly A. inopinatus, particularly in Central European mountain ranges. By relating these range shifts to my population genetic results, I identified conservation units primarily in Eastern Europe, that if preserved would maintain high levels of the present-day genetic diversity of A. inopinatus and continue to provide long-term suitable habitat under future climate warming scenarios.rnIn this thesis I show that despite similar present day distributions the underlying demographic histories of the study species are vastly different, which might be due to differing dispersal capabilities and niche plasticity. I present genetic, climatic, and ecological data that can be used to prioritize conservation efforts for cold-adapted freshwater insects in light of future climate change. Overall, this thesis provides a next step in filling the knowledge gap regarding molecular studies of the arctic-alpine invertebrate fauna. However, there is continued need to explore the phenomenon of arctic-alpine disjunctions to help understand the processes of range expansion, regression, and lineage diversification in Europe’s high latitude and high altitude biota.