Effects of different methods of non‐lethal tissue sampling on butterflies

1. We investigated the effects of two methods of non‐lethal tissue sampling on post‐release flight behaviour (short‐term response) and survival (long‐term response) of two butterflies, Pieris rapae and Coenonympha tullia, within the same natural habitat. We applied three treatments: control (no tissue removal), wing clipping, and leg removal. Our study is the first to directly compare the effects of these common sampling methods. 2. We monitored the flight behaviour of the butterflies by following individuals immediately after their release. In 99 behaviour trials of P. rapae and 101 of C. tullia we found no significant differences in proportion of time spent flying or displacement per unit time among treatment groups in either species. 3. We used standard mark–recapture techniques continuously throughout the flight season to compare the survival of individuals. We marked a total of 687 P. rapae and 490 C. tullia butterflies. We found no significant differences in survival among treatments in either species. 4. We detected differences between the sexes in survival in P. rapae and flight behaviour in C. tullia. In addition to indicating differences in ecology between the sexes, these results also suggest that our analyses were sufficiently powerful to detect a significant effect of tissue removal had such an effect existed. 5. Our work is an important addition to the accumulating evidence that these methods of non‐lethal tissue sampling are generally not detrimental. These sampling techniques closely mimic conditions in the wild, as wing wear and leg losses occur naturally.     

A touch of glue to complete bacteriophage assembly: the tail‐to‐head joining protein (THJP) family

Bacteriophage SPP1 is a nanomachine built to infect the bacterium Bacillus subtilis. The phage particle is composed of an icosahedric capsid, which contains the viral DNA, and a long non‐contractile tail. Capsids and tails are produced in infected cells by two distinct morphogenetic pathways. Characterization of the suppressor‐sensitive mutant SPP1sus82 showed that it produces DNA‐filled capsids and tails but is unable to assemble complete virions. Its purified tails have a normal length but lack a narrow ring that tapers the tail end found at the tail‐to‐head interface. The mutant is defective in production of gp17. The gp17 ring is exposed in free tails competent for viral assembly but becomes shielded in the final virion structure. Recombinant gp17 is active in an in vitro assay to stick together capsids and tails present in extracts of SPP1sus82‐infected cells, leading to formation of infectious particles. Gp17 thus plays a fundamental role in the tail‐to‐head joining reaction, the ultimate step of virus particle assembly. This is the conserved function of gp17 and its structurally related proteins like lambda gpU. This family of proteins can also provide fidelity to termination of the tail tube elongation reaction in a subset of phages including coliphage lambda.     

Mating Behavior and Dual‐Purpose Armaments in a Camel Cricket

Sexual selection has often been dichotomized into intersexual and intrasexual components of selection, favoring ornaments and weapons, respectively. Here, we show that a weapon used in male–male combat is sometimes also used in a functionally similar manner for grasping females during mating. The hind legs of adult males of some species of Pristoceuthophilus camel crickets have strongly bent tibia and stout femora with two large conspicuous spines. Here, we show that (1) leg armature is positively allometric, (2) males use these leg modifications when fighting other males, (3) males sometimes use the same leg modifications in the same functional manner for grabbing and holding females for mating, (4) virgin females show more interest in males than do non‐virgin females, and (5) males are more likely to use their leg modifications to grab and hold females when paired with non‐virgin females than when paired with virgin females. We interpret these results as suggesting that males employ an alternative coercive mating strategy when paired with unreceptive females; alternative explanations are also discussed.     

Sexual selection,phenotypic variation,and allometry in genitalic and non‐genitalic traits in the sexually size‐dimorphic stick insect Micrarchus hystriculeus

The mobility hypothesis could explain the evolution of female‐biased size dimorphism if males with a smaller body size and longer legs have an advantage in scramble competition for mates. This hypothesis is tested by performing a selection analysis in the wild on Micrarchus hystriculeus (Westwood) (Phasmatodea), a sexually size dimorphic stick insect endemic to New Zealand. This analysis examined the form and strength of sexual selection on body size, leg lengths (front, mid and hind), and clasper size (a genitalic trait), and also quantified the degree of phenotypic variation and the allometric scaling pattern of these traits. By contrast to the mobility hypothesis, three lines of evidence were found to support significant stabilizing sexual selection on male hind leg length: a significant nonlinear selection gradient, negative static allometry, and a low degree of phenotypic variation. Hind leg length might be under stabilizing selection in males if having average‐sized legs facilitates female mounting or improves a male's ability to achieve the appropriate copulation position. As predicted, a negative allometric scaling pattern and low phenotypic variation of clasper size is suggestive of stabilizing selection and supports the ‘one‐size‐fits‐all’ hypothesis. Opposite to males, the mid and hind leg lengths of females showed positive static allometry. Relatively longer mid and hind leg lengths in larger females might benefit individuals via the better support of their larger abdomens. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 471–484.     

Looping‐out mechanism for resolution of replicative stress at telomeres

Repetitive DNA is prone to replication fork stalling, which can lead to genome instability. Here, we find that replication fork stalling at telomeres leads to the formation of t‐circle‐tails, a new extrachromosomal structure that consists of circular telomeric DNA with a single‐stranded tail. Structurally, the t‐circle‐tail resembles cyclized leading or lagging replication intermediates that are excised from the genome by topoisomerase II‐mediated cleavage. We also show that the DNA damage repair machinery NHEJ is required for the formation of t‐circle‐tails and for the resolution of stalled replication forks, suggesting that NHEJ, which is normally constitutively suppressed at telomeres, is activated in the context of replication stress. Inhibition of NHEJ or knockout of DNA‐PKcs impairs telomere replication, leading to multiple‐telomere sites (MTS) and telomere shortening. Collectively, our results support a “looping‐out” mechanism, in which the stalled replication fork is cut out and cyclized to form t‐circle‐tails, and broken DNA is religated. The telomere loss induced by replication stress may serve as a new factor that drives replicative senescence and cell aging.     

Effects of tail autotomy on survival,growth and territory occupation in free‐ranging juvenile geckos (Oedura lesueurii)

Abstract Many animals autotomize their tails to facilitate escape from predators. Although tail autotomy can increase the likelihood of surviving a predatory encounter, it may entail subsequent costs, including reduced growth, loss of energy stores, a reduction in reproductive output, loss of social status and a decreased probability of survival during subsequent encounters with predators. To date, few studies have investigated the potential fitness costs of tail autotomy in natural populations. I investigated whether tail loss influenced survival, growth and territory occupation of juvenile velvet geckos Oedura lesueurii in a population where predatory snakes were common. During the 3‐year mark–recapture study, 32% of juveniles voluntarily autotomized their tails when first captured. Analysis of survival using the program mark showed that voluntary tail autotomy did not influence the subsequent survival of juvenile geckos. Survival was age‐dependent and was higher in 1‐year‐old animals (0.98) than in hatchlings (0.76), whereas recapture probabilities were time‐dependent. Growth rates of tailed and tailless juveniles were very similar, but tailless geckos had slow rates of tail regeneration (0.14 mm day⁻¹). Tail autotomy did not influence rock usage by geckos, and both tailed and tailless juveniles used few rocks as diurnal retreat sites (means of 1.64 and 1.47 rocks, respectively) and spent long time periods (85 and 82 days) under the same rocks. Site fidelity may confer survival advantages to juveniles in populations sympatric with ambush foraging snakes. My results show that two potential fitness costs of tail autotomy – decreased growth rates and a lower probability of survival – did not occur in juveniles from this population. However, compared with juveniles, significantly fewer adult geckos (17%) voluntarily autotomized their tails during capture. Because adults possess large tails that are used for lipid storage, the energetic costs of tail autotomy are likely to be much higher in adult than in juvenile O. lesueurii.     

Sexual size dimorphism and assortative mating for morphological traits in Passer domesticus

In this study, assortative mating for different morphological traits was studied in a captive population of house sparrows (Passer domesticus). Males were larger than females. Assortative mating was found for tail length, wing length and general body size. Males with larger badge size mated with females with longer tails. The strongest assortative mating occurred for tail length (r=0.77), and this assortative mating remained significant after controlling for wing length, mass and tarsus length, suggesting that it was not an artefact of assortative mating for body size. The possibility of sexual selection for tail length in the house sparrow is discussed.     

Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Experimental manipulation reveals a trade‐off between weapons and testes

Theory predicts a trade‐off between sexually selected weapons used to secure mates and post‐copulatory traits used to maximize fertilization success. However, individuals that have a greater capacity to acquire resources from the environment may invest more in both pre‐ and post‐copulatory traits, and trade‐offs may not be readily apparent. Here, we manipulate the phenotype of developing individuals to examine allocation trade‐offs between weapons and testes in Mictis profana (Hemiptera: Coreidae), a species where the hind legs are sexually selected weapons used in contests over access to females. We experimentally prevented males from developing weapons by inducing them to autotomize their hind legs before the final moult to adulthood. We compared trait expression in this group to males where autotomy was induced in the mid‐legs, which are presumably not under sexual selection to the same extent. We found males without weapons invested proportionally more in testes mass than those with their mid‐legs removed. Males that developed to adulthood without weapons did not differ from the mid‐leg removal group in other traits potentially under precopulatory sexual selection, other post‐copulatory traits or naturally selected traits. In addition, a sample of adult males from the same population in the wild revealed a positive correlation between investment in testes and weapons. Our study presents a critical contribution to a growing body of literature suggesting the allocation of resources to pre‐ and post‐copulatory sexual traits is influenced by a resource allocation trade‐off and that this trade‐off may only be revealed with experimental manipulation.     

No evidence for size‐assortative mating in the wild despite mutual mate choice in sex‐role‐reversed pipefishes

Size‐assortative mating is a nonrandom association of body size between members of mating pairs and is expected to be common in species with mutual preferences for body size. In this study, we investigated whether there is direct evidence for size‐assortative mating in two species of pipefishes, Syngnathus floridae and S. typhle, that share the characteristics of male pregnancy, sex‐role reversal, and a polygynandrous mating system. We take advantage of microsatellite‐based “genetic‐capture” techniques to match wild‐caught females with female genotypes reconstructed from broods of pregnant males and use these data to explore patterns of size‐assortative mating in these species. We also develop a simulation model to explore how positive, negative, and antagonistic preferences of each sex for body size affect size‐assortative mating. Contrary to expectations, we were unable to find any evidence of size‐assortative mating in either species at different geographic locations or at different sampling times. Furthermore, two traits that potentially confer a fitness advantage in terms of reproductive success, female mating order and number of eggs transferred per female, do not affect pairing patterns in the wild. Results from model simulations demonstrate that strong mating preferences are unlikely to explain the observed patterns of mating in the studied populations. Our study shows that individual mating preferences, as ascertained by laboratory‐based mating trials, can be decoupled from realized patterns of mating in the wild, and therefore, field studies are also necessary to determine actual patterns of mate choice in nature. We conclude that this disconnect between preferences and assortative mating is likely due to ecological constraints and multiple mating that may limit mate choice in natural populations.     

The C‐terminal tails of heterotrimeric kinesin‐2 motor subunits directly bind to α‐tubulin1: Possible implications for cilia‐specific tubulin entry

The assembly of microtubule‐based cytoskeleton propels the cilia and flagella growth. Previous studies have indicated that the kinesin‐2 family motors transport tubulin into the cilia through intraflagellar transport. Here, we report a direct interaction between the C‐terminal tail fragments of heterotrimeric kinesin‐2 and α‐tubulin1 isoforms in vitro. Blot overlay screen, affinity purification from tissue extracts, cosedimentation with subtilisin‐treated microtubule and LC‐ESI‐MS/MS characterization of the tail‐fragment‐associated tubulin identified an association between the tail domains and α‐tubulin1A/D isotype. The interaction was confirmed by Forster's resonance energy transfer assay in tissue‐cultured cells. The overexpression of the recombinant tails in NIH3T3 cells affected the primary cilia growth, which was rescued by coexpression of a α‐tubulin1 transgene. Furthermore, fluorescent recovery after photobleach analysis in the olfactory cilia of Drosophila indicated that tubulin is transported in a non‐particulate form requiring kinesin‐2. These results provide additional new insight into the mechanisms underlying selective tubulin isoform enrichment in the cilia.      

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Distribution and at‐sea behavior of Bermudan White‐tailed Tropicbirds (Phaethon lepturus catesbyi) during the non‐breeding season

The movements and behavior of many taxa of seabirds during the non‐breeding season remain poorly known. For example, although studies conducted in the Pacific and Indian oceans suggest that White‐tailed Tropicbirds (Phaethon lepturus) seldom fly more than a few thousand kilometers from nest colonies after breeding, little is known about the post‐breeding movements and behavior of a subspecies of White‐tailed Tropicbirds (P. l. catesbyi) that breeds on islands in the North Atlantic Ocean. Our objective, therefore, was to use light‐based geolocators to identify the ranges and pelagic activities of White‐tailed Tropicbirds from Bermuda during the non‐breeding periods in 2014–2015 (N = 25) and 2015–2016 (N = 16). Locations were estimated based on changes in light intensity across time, and pelagic activities were determined based on whether geolocators attached to leg bands were wet (i.e., birds resting on the water's surface) or dry (i.e., birds in flight). In 2014, birds spent late summer (July–September) near Bermuda and the British Virgin Islands; by mid‐September, most (N = 17; 68%) birds took a direct easterly route to the Sargasso Sea. In 2015, most post‐breeders (N = 15; 94%) flew east from Bermuda and to the Sargasso before the end of late summer. For both years combined, fall and winter (October–February) ranges extended as far west as North Carolina and as far east as the mid‐Atlantic Ridge. In both years, all birds were located between Bermuda and the British Virgin Islands during the spring (April–May). All birds then flew north to Bermuda in both years, with variations in timing, during April and May. We also found extensive overlap in the ranges of males and females during the non‐breeding season in both years. During the non‐breeding season, White‐tailed Tropicbirds spent 5% of night periods and 41% of day periods in flight in 2014; in 2015, birds spent 8% and 42% of night and day periods, respectively, in flight. Tropicbirds spent more time flying during the day because they hunt by day, detecting prey on the wing by sight. Overall, our results suggest that White‐tailed Tropicbirds that breed in Bermuda are diurnal, nomadic wanderers that range over an extensive area of the Atlantic Ocean during the non‐breeding season.     

The use of a non‐invasive tool for capture–recapture studies on a seahorse Hippocampus guttulatus population

In this study, the spot pattern in Hippocampus guttulatus was analysed using a computer programme algorithm that allowed individual comparison. This methodology was first tested in a controlled environment using 51 adult and 55 juvenile H. guttulatus. Positive matches were obtained in 86·3 and 83·6% of the adults and juveniles, respectively. In a second experiment, monthly surveys were carried out in five selected locations in the Ria Formosa Lagoon, south Portugal, over the course of a year and a total of 980 photographs were analysed. Photographed H. guttulatus were re‐sighted one to nine times during the course of the survey period with an overall re‐sight record of over 30%. Photo‐identification was therefore shown to be a useful tool for non‐invasive mark–recapture studies that can be successfully used to survey the population abundance of H. guttulatus aged 6 months or older in consecutive years. This could be of great value when considering the assessment of H. guttulatus populations and understanding changes over time.     

Updated checklist and DNA barcode‐based species delimitations reveal taxonomic uncertainties among freshwater fishes from the mid‐north‐eastern Caatinga ecoregion,north‐eastern Brazil

The mid‐north‐eastern Caatinga is a semiarid freshwater ecoregion in North‐eastern Brazil that is dominated by temporary rivers and is currently classified as one of the least ichthyologically‐known ecoregions in the world. The present study aimed to provide an updated checklist of mid‐north‐eastern Caatinga ecoregion (MNCE) freshwater fish species and evaluate their taxonomic identity using morphology, DNA barcoding and multiple species delimitation approaches. After reviewing published studies and ichthyological collections, 119 species were identified. Among these were 94 putatively valid native and 14 non‐native species, five undescribed native species, four new records for the MNCE, 11 potential cases of misidentification and 14 species listed as inquirenda. Additionally, 252 individuals from 49 species were barcoded, revealing three potential taxonomic synonyms. The combined molecular approaches estimated a total of 91 native species, although a finalized species list for the MNCE awaits additional taxonomic revisions and field surveys. This study provides the most up‐to‐date species checklist for the MNCE and a molecular reference database for identifying MNCE fishes with DNA barcodes. Results highlight the need to integrate traditional taxonomy with molecular approaches to correctly identify species, especially in taxonomically problematic ecoregions such as the MNCE.     

Plasticity in courtship and sneaking behaviors depending on tail length in the male guppy, <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

Male guppies Poecilia reticulata exhibit two types of mating behavior, i.e., courtship displays for cooperative copulation and sneaking attempts for forced copulation. The frequencies of the two male mating behaviors are influenced by tail length. Males possessing long tails exhibit courtship displays less frequently and sneaking attempts more frequently than those possessing short tails, even though they have similar total lengths. To examine whether these male behavioral tendencies depending on tail length are genetically controlled or are determined by tail length per se, tail length manipulation was conducted. The tail lengths of males that had previously possessed longer tails were surgically shortened to a greater degree than those of their counterparts that had previously possessed shorter tails. Although the frequencies of the mating behaviors exhibited by the latter males did not apparently change, the former males clearly increased the frequency of courtship displays and decreased that of sneaking attempts following tail shortening. These results indicate that males adjust the frequencies of the two mating behaviors according to their tail length. Since females avoid cooperative mating with males possessing long tails, the change in mating behavioral patterns by males depending on their tail length may increase their mating opportunities.     

Potent inhibitors of human matriptase‐1 based on the scaffold of sunflower trypsin inhibitor

Sunflower trypsin inhibitor‐1 (SFTI‐1), a bicyclic tetradecapeptide, has become a versatile tool as a scaffold for the development of the inhibitors of therapeutically relevant serine proteases, among them matriptase and kallikreins. Herein, we report the rational design of potent monocyclic and bicyclic inhibitors of human matriptase‐1. We found that the presence of positive charge and lack of bulky residues at the peptide N‐terminus is required for the maintenance of inhibitory activity. Replacement of the N‐terminal glycine residue by lysine allowed for the chemical conjugation with a fluorophor via the ε‐amino group without significant loss of inhibitory activity. Head‐to‐tail and side‐chain‐to‐tail cyclization resulted in potent inhibitors with comparable activities against matriptase‐1. The most potent synthetic bicyclic inhibitor found in this study (K_i = 2.6 nM at pH 7.6) is a truncated version of SFTI‐1 (cyclo‐KRCTKSIPPRCH) lacking a C‐terminal proline and aspartate residue. It combines an internal disulfide bond with a peptide macrocycle that is formed through side‐chain‐to‐tail cyclization of the ε‐amino group of an N‐terminal lysine and a C‐terminal proline. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Cross‐sex genetic covariances limit the evolvability of wing‐shape within and among species of Drosophila

The independent evolution of males and females is potentially constrained by both sexes inheriting the same alleles from their parents. This genetic constraint can limit the evolvability of complex traits; however, there are few studies of multivariate evolution that incorporate cross‐sex genetic covariances in their predictions. Drosophila wing‐shape has emerged as a model high‐dimensional phenotype; wing‐shape is highly evolvable in contemporary populations, and yet perplexingly stable across phylogenetic timescales. Here, we show that cross‐sex covariances in Drosophila melanogaster, given by the B ‐matrix, may considerably bias wing‐shape evolution. Using random skewers, we show that B would constrain the response to antagonistic selection by 90%, on average, but would double the response to concordant selection. Both cross‐sex within‐trait and cross‐sex cross‐trait covariances determined the predicted response to antagonistic selection, but only cross‐sex within‐trait covariances facilitated the predicted response to concordant selection. Similar patterns were observed in the direction of extant sexual dimorphism in D. melanogaster, and in directions of most and least dimorphic variation across the Drosophila phylogeny. Our results highlight the importance of considering between‐sex genetic covariances when making predictions about evolution on both macro‐ and microevolutionary timescales, and may provide one more explanatory piece in the puzzle of stasis.