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DNA damage in preserved specimens and tissue samples: a molecular assessment
The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage.Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents – such as light, oxygen or formaldehyde – on free nucleotides.We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS.We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.
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Parasegmental appendage allocation in annelids and arthropods and the homology of parapodia and arthropodia
The new animal phylogeny disrupts the traditional taxon Articulata (uniting arthropods and annelids) and thus calls into question the homology of the body segments and appendages in the two groups. Recent work in the annelid Platynereis dumerilii has shown that although the set of genes involved in body segmentation is similar in the two groups, the body units of annelids correspond to arthropod parasegments not segments. This challenges traditional ideas about the homology of "segmental" organs in annelids and arthropods, including their appendages. Here I use the expression of engrailed, wingless and Distal-less in the arthropod Artemia franciscana to identify the parasegment boundary and the appendage primordia. I show that the early body organization including the appendage primordia is parasegmental and thus identical to the annelid organization and by deriving the different adult appendages from a common ground plan I suggest that annelid and arthropod appendages are homologous structures despite their different positions in the adult animals. This also has implications for the new animal phylogeny, because it suggests that Urprotostomia was not only parasegmented but also had parasegmental appendages similar to extant annelids, and that limb-less forms in the Protostomia are derived from limb-bearing forms.
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Tropical parabiotic ants: Highly unusual cuticular substances and low interspecific discrimination
Background:
Associations between animal species require that at least one of the species recognizes its partner. Parabioses are associations of two ant species which co-inhabit the same nest. Ants usually possess an elaborate nestmate recognition system, which is based on cuticular hydrocarbons and allows them to distinguish nestmates from non-nestmates through quantitative or qualitative differences in the hydrocarbon composition. Hence, living in a parabiotic association probably necessitates changes of the nestmate recognition system in both species, since heterospecific ants have to be accepted as nestmates.
Results:
In the present study we report highly unusual cuticular profiles in the parabiotic species Crematogaster modiglianii and Camponotus rufifemur from the tropical rainforest of Borneo. The cuticle of both species is covered by a set of steroids, which are highly unusual surface compounds. They also occur in the Dufour gland of Crematogaster modiglianii in high quantities. The composition of these steroids differed between colonies but was highly similar among the two species of a parabiotic nest. In contrast, hydrocarbon composition of Cr. modiglianii and Ca. rufifemur differed strongly and only overlapped in three regularly occurring and three trace compounds. The hydrocarbon profile of Camponotus rufifemur consisted almost exclusively of methyl-branched alkenes of unusually high chain lengths (up to C49). This species occurred in two sympatric, chemically distinct varieties with almost no hydrocarbons in common. Cr. modiglianii discriminated between these two varieties. It only tolerated workers of the Ca. rufifemur variety it was associated with, but attacked the respective others. However, Cr. modiglianii did not distinguish its own Ca. rufifemur partner from allocolonial Ca. rufifemur workers of the same variety.
Conclusion:
We conclude that there is a mutual substance transfer between Cr. modiglianii and Ca. rufifemur. Ca. rufifemur actively or passively acquires cuticular steroids from its Cr. modiglianii partner, while the latter acquires at least two cuticular hydrocarbons from Ca. rufifemur. The cuticular substances of both species are highly unusual regarding both substance classes and chain lengths, which may cause the apparent inability of Cr. modiglianii to discriminate Ca. rufifemur nestmates from allocolonial Ca. rufifemur workers of the same chemical variety.
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Termites, hemimetabolous diploid white ants?
Ants and termites are the most abundant animals on earth. Their ecological success is attributed to their social life. They live in colonies consisting of few reproducing individuals, while the large majority of colony members (workers/soldiers) forego reproduction at least temporarilly. Despite their apparent resemblance in social organisation, both groups evolved social life independently. Termites are basically social cockroaches, while ants evolved from predatory wasps. In this review, I will concentrate on termites with an ancestral life type, the wood-dwelling termites, to compare them with ants. Their different ancestries provided both groups with different life history pre-adaptations for social evolution. Like their closest relatives, the woodroaches, wood-dwelling termites live inside their food, a piece of wood. Thus, intensive costly food provisioning of their young is not necessary, especially as young instars are rather independent due to their hemimetabolous development. In contrast, ants are progressive food provisioners which have to care intensively for their helpless brood. Corresponding to the precocial – altricial analogy, helping by workers is selected in ants, while new evidence suggests that wood-dwelling termite workers are less engaged in brood care. Rather they seem to stay in the nest because there is generally low selection for dispersal. The nest presents a safe haven with no local resource competition as long as food is abundant (which is generally the case), while founding a new colony is very risky. Despite these differences between ants and termites, their common dwelling life style resulted in convergent evolution, especially winglessness, that probably accounts for the striking similarity between both groups. In ants, all workers are wingless and winglessness in sexuals evolved in several taxa as a derived trait. In wood-dwelling termites, workers are by default wingless as they are immatures. These immatures can develop into winged sexuals that disperse and found a new nest or into neotenic replacement reproductives that inherit the natal colony. Depending on the worker instar from which the latter develop, the neotenic reproductives are either apterous or brachypterous, but never winged. I propose that this wing polyphenism might present a basis for the evolution of social life in termites.
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Myogenesis in the basal bilaterian Symsagittifera roscoffensis (Acoela)
Background:
In order to increase the weak database concerning the organogenesis of Acoela – a clade regarded by many as the earliest extant offshoot of Bilateria and thus of particular interest for studies concerning the evolution of animal bodyplans – we analyzed the development of the musculature of Symsagittifera roscoffensis using F-actin labelling, confocal laserscanning microscopy, and 3D reconstruction software.
Results:
At 40% of development between egg deposition and hatching short subepidermal fibres form. Muscle fibre development in the anterior body half precedes myogenesis in the posterior half. At 42% of development a grid of outer circular and inner longitudinal muscles is present in the bodywall. New circular muscles either branch off from present fibres or form adjacent to existing ones. The number of circular muscles is higher than that of the longitudinal muscles throughout all life cycle stages. Diagonal, circular and longitudinal muscles are initially rare but their number increases with time. The ventral side bears U-shaped muscles around the mouth, which in addition is surrounded by a sphincter muscle. With the exception of the region of the statocyst, dorsoventral muscles are present along the entire body of juveniles and adults, while adults additionally exhibit radially oriented internal muscles in the anterior tip. Outer diagonal muscles are present at the dorsal anterior tip of the adult. In adult animals, the male gonopore with its associated sexual organs expresses distinct muscles. No specific statocyst muscles were found. The muscle mantles of the needle-shaped sagittocysts are situated along the lateral edges of the animal and in the posterior end close to the male gonopore. In both juveniles and adults, non-muscular filaments, which stain positively for F-actin, are associated with certain sensory cells outside the bodywall musculature.
Conclusion:
Compared to the myoanatomy of other acoel taxa, Symsagittifera roscoffensis shows a very complex musculature. Although data on presumably basal acoel clades are still scarce, the information currently available suggests an elaborated musculature with longitudinal, circular and U-shaped muscles as being part of the ancestral acoel bodyplan, thus increasing the possibility that Urbilateria likewise had a relatively complicated muscular ground pattern.
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Vocal mother-offspring communication in guinea pigs: females adjust maternal responsiveness to litter size
Background:
In parent-offspring communication, vocal signals are often used to attract attention and offspring might call to induce parental behaviour. In guinea pigs (Cavia aperea f. porcellus) mothers wean larger litters later than small ones, but it is unknown whether this difference depends on processes induced during pregnancy or is influenced post-natally by the number of pups present. We here tested with playback-experiments using pup separation calls whether mothers with cross-fostered large experimental litters (four-pup-litters) were more responsive to offspring calls and maintained responsiveness for longer than mothers with small experimental litters (two-pup-litters). Mothers were tested when two pups were suckling i.e. when both teats were occupied.
Results:
Mothers of four-pup litters responded stronger to broadcast pup separation calls than those with two-pup litters. Additionally, we tested the mothers' responsiveness to pup separation calls in the absence of their pups on day 8 and 20 of lactation. Mothers of four-pup litters responded stronger and showed no decrease in responsiveness from day 8 to 20, whereas mothers of two-pup litters responded less and decreased responsiveness from day 8 to 20. Mothers of four-pup litters also weaned their pups 5 days later than those of two-pup litters.
Conclusion:
Measured by their response to pup calls and by time to weaning, guinea pig mothers adjust maternal responsiveness to litter size. This behaviour is likely to be an adaptive strategy in resource allocation during reproduction.
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Blocking primers to enhance PCR amplification of rare sequences in mixed samples – a case study on prey DNA in Antarctic krill stomachs
Background:
Identification of DNA sequence diversity is a powerful means for assessing the species present in environmental samples. The most common molecular strategies for estimating taxonomic composition depend upon PCR with universal primers that amplify an orthologous DNA region from a range of species. The diversity of sequences within a sample that can be detected by universal primers is often compromised by high concentrations of some DNA templates. If the DNA within the sample contains a small number of sequences in relatively high concentrations, then less concentrated sequences are often not amplified because the PCR favours the dominant DNA types. This is a particular problem in molecular diet studies, where predator DNA is often present in great excess of food-derived DNA.
Results:
We have developed a strategy where a universal PCR simultaneously amplifies DNA from food items present in DNA purified from stomach samples, while the predator's own DNA is blocked from amplification by the addition of a modified predator-specific blocking primer. Three different types of modified primers were tested out; one annealing inhibiting primer overlapping with the 3' end of one of the universal primers, another annealing inhibiting primer also having an internal modification of five dI molecules making it a dual priming oligo, and a third elongation arrest primer located between the two universal primers. All blocking primers were modified with a C3 spacer. In artificial PCR mixtures, annealing inhibiting primers proved to be the most efficient ones and this method reduced predator amplicons to undetectable levels even when predator template was present in 1000 fold excess of the prey template. The prey template then showed strong PCR amplification where none was detectable without the addition of blocking primer. Our method was applied to identifying the winter food of one of the most abundant animals in the world, the Antarctic krill, Euphausia superba. Dietary item DNA was PCR amplified from a range of species in krill stomachs for which we had no prior sequence knowledge.
Conclusion:
We present a simple, robust and cheap method that is easily adaptable to many situations where a rare DNA template is to be PCR amplified in the presence of a higher concentration template with identical PCR primer binding sites.
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Outlining eicosanoid biosynthesis in the crustacean Daphnia
Background:
Eicosanoids are biologically active, oxygenated metabolites of three C20 polyunsaturated fatty acids. They act as signalling molecules within the autocrine or paracrine system in both vertebrates and invertebrates mainly functioning as important mediators in reproduction, the immune system and ion transport. The biosynthesis of eicosanoids has been intensively studied in mammals and it is known that they are synthesised from the fatty acid, arachidonic acid, through either the cyclooxygenase (COX) pathway; the lipoxygenase (LOX) pathway; or the cytochrome P450 epoxygenase pathway. However, little is still known about the synthesis and structure of the pathway in invertebrates.
Results:
Here, we show transcriptomic evidence from Daphnia magna (Crustacea: Branchiopoda) together with a bioinformatic analysis of the D. pulex genome providing insight on the role of eicosanoids in these crustaceans as well as outlining a putative pathway of eicosanoid biosynthesis. Daphnia appear only to have one copy of the gene encoding the key enzyme COX, and phylogenetic analysis reveals that the predicted protein sequence of Daphnia COX clusters with other invertebrates. There is no current evidence of an epoxygenase pathway in Daphnia; however, LOX products are most certainly synthesised in daphnids.
Conclusion:
We have outlined the structure of eicosanoid biosynthesis in Daphnia, a key genus in freshwater ecosystems. Improved knowledge of the function and synthesis of eicosanoids in Daphnia and other invertebrates could have important implications for several areas within ecology. This provisional overview of daphnid eicosanoid biosynthesis provides a guide on where to focus future research activities in this area.
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Adult nutrition and butterfly fitness: effects of diet quality on reproductive output, egg composition, and egg hatching success
Background:
In the Lepidoptera it was historically believed that adult butterflies rely primarily on larval-derived nutrients for reproduction and somatic maintenance. However, recent studies highlight the complex interactions between storage reserves and adult income, and that the latter may contribute significantly to reproduction. Effects of adult diet were commonly assessed by determining the number and/or size of the eggs produced, whilst its consequences for egg composition and offspring viability were largely neglected (as is generally true for insects). We here specifically focus on these latter issues by using the fruit-feeding tropical butterfly Bicyclus anynana, which is highly dependent on adult-derived carbohydrates for reproduction.
Results:
Adult diet of female B. anynana had pronounced effects on fecundity, egg composition and egg hatching success, with butterflies feeding on the complex nutrition of banana fruit performing best. Adding vitamins and minerals to a sucrose-based diet increased fecundity, but not offspring viability. All other groups (plain sucrose solution, sucrose solution enriched with lipids or yeast) had a substantially lower fecundity and egg hatching success compared to the banana group. Differences were particularly pronounced later in life, presumably indicating the depletion of essential nutrients in sucrose-fed females. Effects of adult diet on egg composition were not straightforward, indicating complex interactions among specific compounds. There was some evidence that total egg energy and water content were related to hatching success, while egg protein, lipid, glycogen and free carbohydrate content did not seem to limit successful development.
Conclusion:
The patterns shown here exemplify the complexity of reproductive resource allocation in B. anynana, and the need to consider egg composition and offspring viability when trying to estimate the effects of adult nutrition on fitness in this butterfly and other insects.
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Development of the retinotectal system in the direct-developing frog Eleutherodactylus coqui in comparison with other anurans
Background:
Frogs primitively have a biphasic life history with an aquatic larva (tadpole) and a usually terrestrial adult. However, direct developing frogs of the genus Eleutherodactylus have lost a free living larval stage. Many larval structures never form during development of Eleutherodactylus, while limbs, spinal cord, and an adult-like cranial musculoskeletal system develop precociously.
Results:
Here, I compare growth and differentiation of the retina and tectum and development of early axon tracts in the brain between Eleutherodactylus coqui and the biphasically developing frogs Discoglossus pictus, Physalaemus pustulosus, and Xenopus laevis using morphometry, immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and acetylated tubulin, biocytin tracing, and in situ hybridization for NeuroD. Findings of the present study indicate that retinotectal development was greatly altered during evolution of Eleutherodactlyus mostly due to acceleration of cell proliferation and growth in retina and tectum. However, differentiation of retina, tectum, and fiber tracts in the embryonic brain proceed along a conserved slower schedule and remain temporally coordinated with each other in E. coqui.
Conclusion:
These findings reveal a mosaic pattern of changes in the development of the central nervous system (CNS) during evolution of the direct developing genus Eleutherodactylus. Whereas differentiation events in directly interconnected parts of the CNS such as retina, tectum, and brain tracts remained coordinated presumably due to their interdependent development, they were dissociated from proliferation control and from differentiation events in other parts of the CNS such as the spinal cord. This suggests that mosaic evolutionary changes reflect the modular character of CNS development.
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