Reproductive plasticity in <Emphasis Type="Italic">Petrolisthes armatus</Emphasis> (Anomura,Porcellanidae): a comparison between a Pacific and an Atlantic population

The porcellanid crab Petrolisthes armatus, with a known geographic distribution covering a wide range of latitudes, was selected to conduct a comparative study on egg production and reproductive output between two populations from Pacific and Atlantic coasts. Ovigerous females were collected between September and November 2005 in Punta Morales, Pacific coast of Costa Rica (n = 137) and from March 2005 to July 2006 in Araçá region, São Paulo, Brazil (n = 46). The mean size of females was statistically different between both populations with larger females from Brazil than Costa Rica (7.9 ± 1.31 and 6.6 ± 0.95 mm CW, respectively). Likewise, egg production was significantly different in both populations and started in Pacific Costa Rica at a smaller female size than in Brazil. Specimens from Pacific Costa Rica produced roughly three times more eggs than equally sized females from Brazil, while newly extruded embryos were larger in Brazil than in Costa Rica (0.045 and 0.039 mm³, respectively). Egg volume increased during embryogenesis by 112.8 and 164.5% in Costa Rican and Brazilian populations, respectively. The egg water content increased steadily in both populations; however, eggs produced in Brazil contained always more water than those from the same developmental stage in Costa Rica. Average reproductive output (based on dry weight) was substantially lower in Brazil (0.031) than in Costa Rica (0.065). Our results confirm an impressive intraspecific plasticity of reproductive features in an intertidal decapod. The observed phenotypical variability might be related to local environmental conditions as well as to the location of the studied population concerning its geographic range of distribution.     

Pollen feeding in Heliconius butterflies: the singular evolution of an adaptive suite

Major evolutionary transitions can be triggered by behavioural novelty, and are often associated with ‘adaptive suites’, which involve shifts in multiple co-adapted traits subject to complex interactions. Heliconius butterflies represent one such example, actively feeding on pollen, a behaviour unique among butterflies. Pollen feeding permits a prolonged reproductive lifespan, and co-occurs with a constellation of behavioural, neuroanatomical, life history, morphological and physiological traits that are absent in closely related, non-pollen-feeding genera. As a highly tractable system, supported by considerable ecological and genomic data, Heliconius are an excellent model for investigating how behavioural innovation can trigger a cascade of adaptive shifts in multiple diverse, but interrelated, traits. Here, we synthesize current knowledge of pollen feeding in Heliconius, and explore potential interactions between associated, putatively adaptive, traits. Currently, no physiological, morphological or molecular innovation has been explicitly linked to the origin of pollen feeding, and several hypothesized links between different aspects of Heliconius biology remain poorly tested. However, resolving these uncertainties will contribute to our understanding of how behavioural innovations evolve and subsequently alter the evolutionary trajectories of diverse traits impacting resource acquisition, life history, senescence and cognition.     

Evidence of protease in the saliva of the butterfly Heliconius melpomene (L.) (Nymphalidae, Lepidoptera)

Butterflies of the genus Heliconius are well known for their peculiar habits of utilizing pollen as a source of amino acids. Saliva plays a major role in the process of extracting amino acids and proteins from the pollen grains. In this investigation, we obtained samples of saliva from adult Heliconius melpomene by placing pumpkin pollen or fine glass-beads on the proboscis, which stimulates the butterflies to release saliva. Proteolytic activity was determined in the saliva by an insoluble protein-dye that turns blue when cleaved by proteases. Its extinction value was measured with a spectrophotometer at 595 nm. Both the saliva sampled with pollen and the saliva obtained from inert glass-beads exhibit proteolytic activity demonstrating that the saliva contains proteases. The proteolytic activity of the pollen/saliva samples was higher than that of the glass-bead/saliva samples, which we attribute to the stimulating effects of pollen, such as taste, smell, and texture, and not to proteases which might have been liberated from the pollen. This is indicated by the fact that pollen samples without saliva showed only a negligible indication for proteolytic activity. In general, females exhibit higher proteolytic activities than males, presumably due to their greater amino acid investment in reproduction. We present here first evidence for the existence of proteases in the saliva of a butterfly species and suggest that these enzymes are crucial for the use of amino acids and proteins from pollen in Heliconius butterflies.     

Pollen feeding proteomics: Salivary proteins of the passion flower butterfly,Heliconius melpomene

While most adult Lepidoptera use flower nectar as their primary food source, butterflies in the genus Heliconius have evolved the novel ability to acquire amino acids from consuming pollen. Heliconius butterflies collect pollen on their proboscis, moisten the pollen with saliva, and use a combination of mechanical disruption and chemical degradation to release free amino acids that are subsequently re-ingested in the saliva. Little is known about the molecular mechanisms of this complex pollen feeding adaptation. Here we report an initial shotgun proteomic analysis of saliva from Heliconius melpomene. Results from liquid-chromatography tandem mass-spectrometry confidently identified 31 salivary proteins, most of which contained predicted signal peptides, consistent with extracellular secretion. Further bioinformatic annotation of these salivary proteins indicated the presence of four distinct functional classes: proteolysis (10 proteins), carbohydrate hydrolysis (5), immunity (6), and “housekeeping” (4). Additionally, six proteins could not be functionally annotated beyond containing a predicted signal sequence. The presence of several salivary proteases is consistent with previous demonstrations that Heliconius saliva has proteolytic capacity. It is likely that these proteins play a key role in generating free amino acids during pollen digestion. The identification of proteins functioning in carbohydrate hydrolysis is consistent with Heliconius butterflies consuming nectar, like other lepidopterans, as well as pollen. Immune-related proteins in saliva are also expected, given that ingestion of pathogens is a likely route to infection. The few “housekeeping” proteins are likely not true salivary proteins and reflect a modest level of contamination that occurred during saliva collection. Among the unannotated proteins were two sets of paralogs, each seemingly the result of a relatively recent tandem duplication. These results offer a first glimpse into the molecular foundation of Heliconius pollen feeding and provide a substantial advance towards comprehensively understanding this striking evolutionary novelty.     

Behavioral Adaptations to Pollen-Feeding in Heliconius Butterflies (Nymphalidae, Heliconiinae): An Experiment Using Lantana Flowers

Butterflies in the genera Heliconius and Laparus obtain fitness-related benefits from using amino acids derived from pollen. These butterflies have morphological features of the proboscis that facilitate pollen-feeding. Here we investigate behavioral characteristics potentially involved in pollen-gathering. Analysis of four behavioral characters showed that pollen-feeding species manipulate Lantana flowers faster and more thoroughly compared to non-pollen-feeding relatives. Although this suggests that pollen-feeding species are potentially more efficient in harvesting pollen, every butterfly tested successfully removed pollen from Lantana and non-pollen-feeding butterflies generally extracted larger amounts of pollen than Heliconius and Laparus. Morphological characteristics of the proboscis and the production of abundant fluid exudate help keep pollen attached in the proboscis for long periods of time—possibly key to Heliconius' and Laparus' ability to obtain amino acids from pollen. Our results, in concert with those of previous morphological analysis, indicate that behavioral and structural attributes associated with pollen-feeding in Heliconius and Laparus are subtle modifications of widespread butterfly characteristics and raise the question why other butterflies do not use pollen in their diet.     

Pollen production and release in <Emphasis Type="Italic">Mesua ferrea</Emphasis> Linn. (Guttiferae): a spatio-temporal pattern

Based on a five-year study of pollen production and release in two different natural populations of Mesua ferrea from Indo-Burma region of Northeast India, we determined that pollen output follows a spatio-temporal pattern. Pollen productivity determinations considered various sources of variability, including the number of flowers per branch, flowers per tree, anthers per tree and pollen grains per tree. Each of these parameters revealed significant year-to-year and population effects. Anthesis follows a forenoon pattern, whereas anther dehiscence pursues the diurnal pattern. The former was significantly correlated with the timing of floral visitation and pollen deposition on stigmas. The latter, however, had significant relationship with the deposition of pollen grains on microscopic slides. The Apis and Xylocopa bees are the efficient pollinators to achieve the reproductive success in M. ferrea. Annual production of pollen per tree varied from averages of 1.07 ± 0.10 × 10¹⁰ and 3.24 ± 0.16 × 10¹⁰ in years of low production, with alternate high years, producing 3.85 ± 0.34 × 10¹⁰ and 8.22 ± 0.76 × 10¹⁰ pollen grains per tree.     

Cell division study in ‘pollen mother cells’ and ‘pollen grains’ of in vivo and ex vitro plants in Drimiopsis botryoides

The various normal and abnormal stages of meiosis and pollen mitosis of Drimiopsis botryoides are described, and a comparison between naturally propagated in vivo and tissue culture derived ex vitro plants in respect to their cytological behaviour presented. We also describe the floral morphology and investigate the relationship between the floral developmental stages and the progression of microgametogenesis. In total, 33 bivalents are observed in diakinesis, which indicate the diploid number 2n = 66 and this number is cross-checked by a haploid set of n = 33 chromosomes in pollen mitosis. Only 6.8% and 4.9% meiotic abnormalities were recorded on in vivo and ex vitro plants, respectively, which led to the formation of non-viable pollen. Finally, the microspores have to develop into tri-cellular male gametophyte. Only 0.2% pollen grains are found with a micro-nucleus. Though the higher pollen viability was recorded on both in vivo (89.3 ± 4.1%) and ex vitro (92.1 ± 4.6%) plants, but surprisingly the pollen germination rate is extremely low with 13.6 ± 1.74% and 21.3 ± 1.55%, respectively. The present study obviously enriches the cytological database of D. botryoides and may help future research on androgenesis and genetic improvement.     

The evolution of adult pollen feeding did not alter postembryonic growth in Heliconius butterflies

For many animals, the availability and provision of dietary resources can vary markedly between juvenile and adult stages, often leading to a temporal separation of nutrient acquisition and use. Juvenile developmental programs are likely limited by the energetic demands of many adult tissues and processes with early developmental origins. Enhanced dietary quality in the adult stage may, therefore, alter selection on life history and growth patterns in juvenile stages. Heliconius are unique among butterflies in actively collecting and digesting pollen grains, which provide an adult source of essential amino acids. The origin of pollen feeding has therefore previously been hypothesized to lift constraints on larval growth rates, allowing Heliconius to spend less time as larvae when they are most vulnerable to predation. By measuring larval and pupal life‐history traits across three pollen‐feeding and three nonpollen‐feeding Heliconiini, we provide the first test of this hypothesis. Although we detect significant interspecific variation in larval and pupal development, we do not find any consistent shift associated with pollen feeding. We discuss how this result may fit with patterns of nitrogen allocation, the benefits of nitrogenous stores, and developmental limitations on growth. Our results provide a framework for studies aiming to link innovations in adult Heliconius to altered selection regimes and developmental programs in early life stages.     

Pollen flow in Psiguria warscewiczii: a comparison of Heliconius butterflies and hummingbirds

Summary Pollen dispersal by male and female Heliconius butterflies and hermit hummingbirds was examined in a natural population of the tropical vine Psiguria warscewiczii. Fluorescent dyes were used to track flowers visited by individual pollinators. Mean distance of pollen dispersal was significantly higher by males than by female Heliconius. Butterflies dispersed pollen greater distances than did hummingbirds. There were no significant differences in dispersal distance among species of Heliconius. The number of flowers or plants to which dye was dispersed was greater for Heliconius than for hummingbirds.The home range behavior and site specificity exhibited by Heliconius, together with the preference by most Heliconius species in the study site for Psiguria led us to examine the relationship between the butterflies' home range and its daily foraging among Psiguria. A Heliconius mark-recapture program was conducted simultaneously with the dye program. This study showed that individual butterflies, for which extensive recaptures were made, visited an area of Psiguria plants on any one day that corresponded to the area in which those butterflies were caught or observed. The area over which a butterfly moved was termed its home range; within home ranges relative movement by butterflies was estimated as the mean distance between sequential captures. This estimate differed significantly among species of Heliconius; males of all species moving greater distances than females.     

Lepanthes variabilis (Orchidaceae: Pleurothallidinae), a new endemic species from Costa Rica

Lepanthes variabilis, a new species from Costa Rica, is described and illustrated. It is endemic to the northern part of the Talamanca mountain range, from 2750–2850 m. It is compared to the similar L. minutilabia, which also occurs in Costa Rica.     

Potential impact of climate change on fungal distributions: analysis of 2 years of contrasting weather in the UK

The impact of climate change on fungal growth and spore production is less well documented than for allergenic pollen grains, although similar implications for respiratory tract diseases in humans occur. Fungal spores are commonly described as either “dry” or “wet” according to the type of weather associated with their occurrence in the air. This study examined the distribution of selected fungal spores (Alternaria spp., Cladosporium spp., Didymella spp., Epicoccum spp., Leptosphaeria spp. and rusts) occurring in the West Midlands of UK during 2 years of contrasting weather. Spore specimens were collected using a 7-day volumetric air sampler and then analysed with the aid of light microscopy. Distributions of spores were then studied using normality tests and Mann–Whitney U test, while relationships with meteorological parameters were investigated using Spearman’s rank test and angular-linear correlation for wind direction analysis. Our results showed that so-called wet spores were more sensitive to the weather changes showing statistically significant differences between the 2 years of study, in contrast to “dry” spores. We predict that in following years we will observe accelerated levels in allergenic fungal spore production as well as changes in species diversity. This study could be a starting point to revise the grouping system of fungal spores as either “dry” or “wet” types and their response to climate change.     

Ovarian dynamics in Heliconius butterflies: Correlations among daily oviposition rates,egg weights,and quantitative aspects of oögenesis

Both oviposition and ovarian morphology were studied in individual butterflies of the neotropical genus Heliconius, which as adults ingest amino acids from pollen, live up to 6 months, and have continuous oögenesis. Among female Heliconius charitonius, daily oviposition correlates directly with the total number of oöcytes developing in the ovaries. The calculated time required for complete growth of one oöcyte is, however, reasonably constant among individuals of a species with widely varying oviposition rates. Thus, within a species, butterflies laying more eggs per day are not necessarily maturing each at a faster rate, but are processing more oöcytes simultaneously in their ovaries. A further correlation between oviposition rate and adult size suggests that in H. charitonius both ovarian capacity and daily egg production are determined ultimately by extent and/or quality of larval nutrition. Among other Heliconius species, those producing larger eggs generally take longer to make them, but also may develop more oöcytes at the same time in their ovaries. Finally, maximal volume attained by the cap of seven nurse cells associated with each growing oöcyte appears constant within a species and among species is directly proportional to average mature egg weight.     

Metamorphosis of a Butterfly-Associated Bacterial Community

Butterflies are charismatic insects that have long been a focus of biological research. They are also habitats for microorganisms, yet these microbial symbionts are little-studied, despite their likely importance to butterfly ecology and evolution. In particular, the diversity and composition of the microbial communities inhabiting adult butterflies remain uncharacterized, and it is unknown how the larval (caterpillar) and adult microbiota compare. To address these knowledge gaps, we used Illumina sequencing of 16S rRNA genes from internal bacterial communities associated with multiple life stages of the neotropical butterfly Heliconius erato. We found that the leaf-chewing larvae and nectar- and pollen-feeding adults of H. erato contain markedly distinct bacterial communities, a pattern presumably rooted in their distinct diets. Larvae and adult butterflies host relatively small and similar numbers of bacterial phylotypes, but few are common to both stages. The larval microbiota clearly simplifies and reorganizes during metamorphosis; thus, structural changes in a butterfly''s bacterial community parallel those in its own morphology. We furthermore identify specific bacterial taxa that may mediate larval and adult feeding biology in Heliconius and other butterflies. Although male and female Heliconius adults differ in reproductive physiology and degree of pollen feeding, bacterial communities associated with H. erato are not sexually dimorphic. Lastly, we show that captive and wild individuals host different microbiota, a finding that may have important implications for the relevance of experimental studies using captive butterflies.     

Production of aborted pollen in marsh species of Chenopodiaceae: evidence of partial male sterility in Suadeaea and Salsoleae species

The main objective of this study is to establish the level of pollen abortion prior to its release from the anther in species of Chenopodiaceae in marsh habitats and to compare this level among the taxons, locations and positions of the flowers in the dichasium and the relative positions of the anthers. We established the level of nonfunctional pollen formation in 39 samples belonging to 10 species, using lactophenol cotton blue staining. Aborted pollen grains were found to be significantly smaller than normal ones. The results revealed differences in the percentage of normal grains among different species and tribes studied: Atripliceae (56.1 ± 27.1%), Salicornieae (80.36 ± 16.52%), Suaedeae (54.7 ± 31.2%) and Salsoleae (32.5 ± 25.7%). In some species there were significant differences among the populations studied, but in species of Saliconieae we found no differences either between different positions in the dicasia or in the anther. In the tribes Suaedeae and Salsoleae, we postulate the existence of a system that maintains different levels of partial male sterility among individuals in the populations, which would reduce the autogamous fruit set rate and favor the cross-pollination rates of sterile male individuals. We base this on high intrapopulation variability at those levels, on the constancy with which they are presented in the different populations studied, and on the lack of interannual differences.     

Pollen morphology of some taxa of Vicia L. subgenus Vicia (Fabaceae) from Turkey

In this study, the pollen morphology of 11 taxa belonging to Atossa (Alef.) Asch. & Graebner, Hypechusa (Alef.) Asch. & Graebner, Peregrinae Kupicha, Wiggersia (Alef.) Maxted, Vicia L. and Narbonensis (Radzhi) Maxted sections of the genus Vicia L. subgenus Vicia (Fabeae, Fabaceae) naturally growing in Turkey has been studied using Light Microscopy (LM) and Scanning Electron Microscopy (SEM) to evaluate the taxonomic relevance of pollen characters. Twelve morphometric characters are analysed with one-way analysis of variance and Tukey’s honestly significant difference test for multiple comparisons. Of the taxa studied V. narbonensis var. narbonensis (sect. Narbonensis) has the largest pollen grains (P = 51.98 μm × E = 30.52 μm) and V. lathyroides (sect. Wiggersia) has the smallest pollen grains (P = 27.71 μm × E = 20.14 μm). The pollen grains are subprolate to perprolate (P/E = 1.16–2.11), but the prolate shape occurs in the majority of the taxa. The regular pollen grains of all taxa are trizonocolporate, isopolar, and released in monads. Ornamentation of the mesocolpium is psilate-perforate in V. lathyroides (sect. Wiggersia), reticulate-rugulate in V. narbonensis var. narbonensis (sect. Narbonensis), (micro)reticulate in V. sericocarpa var. sericocarpa (sect. Hypechusa), V. sativa subsp. sativa (sect. Vicia) and V. grandiflora var. grandiflora (sect. Vicia), and reticulate-perforate in the remaining taxa. The apocolpium and colpus area are psilate or perforate in all taxa except V. sericocarpa var. sericocarpa (sect. Hypechusa) and V. sativa subsp. sativa (sect. Vicia), which exhibit the obscurely reticulate-perforate pattern. Several palynological features have taxonomic importance in sectional level: polar axis, equatorial diameter, pollen shape (P/E ratio), colpus length, colpus width, size of pori, porus length/width ratio, lumina diameter, muri thickness and ornamentation. The results also indicate that pollen characters can be useful in distinguishing the examined taxa.     

Meiotic behavior and pollen morphology variation in Centaurium pulchellum (Gentianaceae)

Centaurium pulchellum is an annual herb native to Europe, but introduced in South America, where it is widely used in the preparation of digestive infusions and bitter drinks. In this species, a wide variation in the aperture pattern of pollen grains was reported and has been attributed to environmental factors or irregularities at meiosis. For this reason, cytological and palynological studies have been undertaken on two different populations. The pollen grain analysis showed that some types are more frequent within each population, but the most common forms were the typical 3-colporate and 4-colporate. The cytological analysis revealed that the analyzed populations of C. pulchellum have chromosome number 2n = 36. The presence of tetravalents at meiosis strongly suggests that these populations are autotetraploid based on x = 9. The meiotic behavior showed a significant percentage of irregularities in different phases: off-plate bivalents, precocious segregation, laggard chromosomes, bridges, and cytomixis. However these irregularities are not related to the variation in the aperture pattern of pollen grains. The heteromorphism in the pollen grains observed in C. pulchellum could be a normal condition to which the species is well adapted.     

Effects of 900 MHz Radiofrequency Radiation on Skin Hydroxyproline Contents

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level (p = 0.049, Mann–Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin.     

Impaired male meiosis, morphology and distribution pattern of different cytotypes of Bupleurum lanceolatum Wall. (Apiaceae) from the Western Himalayas

Detailed male meiosis, critical morphological observations and distribution pattern of diploid as well as tetraploid cytotypes of the Western Himalayan species, Bupleurum lanceolatum have been evaluated at present. A diploid (n = 8) cytotype is reported from Kashmir, whereas, both diploid (n = 8) and tetraploid (n = 16) cytotypes are available from two districts Kangra and Sirmaur of Himachal Pradesh. Out of these, the tetraploid cytotype makes new addition for the species on a worldwide basis. As per behavior, a tetraploid cytotype is characterized by abnormal meiosis leading to high pollen sterility and size variation of the pollen grains. Morphologically, tetraploids are noted to be luxuriant in comparison to the diploids.     

Pollen and ovule production in wind-pollinated species with special reference to Juncus

The reproductive biology of wind-pollinated species in terms of pollen and ovule production is rarely studied compared with zoophilous species, despite available hypotheses on the effect of growth form and life-history traits on reproductive investment. Here, we use published data and new data for species of Juncus and Luzula (Juncaceae) to test the hypotheses that, in wind-pollinated species, woody perennials should exhibit larger pollen–ovule (P/O) ratios than herbaceous species and that species with separate sexes have larger P/O ratios than homoecious species. In total, we report pollen and ovule production for 291 wind-pollinated species, including 19 Juncus and 5 Luzula species. Compared with other wind-pollinated species, Juncus exhibits unusually low P/O ratios (log P/O = 2.06 ± 0.46) because of high ovule production. We argue that the high ovule and seed production in Juncus, associated with frequent self-fertilization, may be beneficial in habitats preferred by the genus. In general, we found higher P/O ratios in woody perennials (log P/O = 4.37 ± 1.18) or in species with separate sexes (log P/O = 4.28 ± 1.12) than in herbaceous (log P/O = 3.51 ± 0.77) or homoecious (log P/O = 3.52 ± 0.80) species, respectively. However, when we analyzed woody perennials separately, we found no significant difference in P/O ratios between homoecious and nonhomoecious species. We argue that woody perennials, independent of dicliny, may be preferentially outcrossed and therefore exhibit decreased variation in mating systems compared with herbs. Because the degree of outcrossing correlates with P/O ratios, differences between homoecious and nonhomoecious woody perennials could be less pronounced.