A non-doubling DNA series in somatic tissues of the locusts Schistocerca gregaria (Forskål) and Locusta migratoria (Linn.)

Locusta migratoria has only 70% of the germ line DNA value of the related species Schistocerca gregaria in spite of a uniform karyotype. This difference is maintained in the nuclei of the testis wall, ovariole tip, mid-gut diverticulum, Malpighian tubule and fat. The distribution of nuclear DNA contents is tissue specific but the peaks in the distributions do not conform with members of a doubling series. It is suggested that both phenomena may be connected with the mechanism of tissue differentiation in insects, the latter by differential replication of those chromosome regions which are active in particular tissues.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

The DNA content of polytene nuclei in midgut and Malpighian tubule cells of adultDrosophila melanogaster

Summary The amounts of DNA in midgut and Malpighian tubule cells of adult maleDrosophila melanogaster have been determined by Feulgen-DNA cytophotometry. The DNA values fall into discrete classes reflecting different levels of polyteny. The maximum level is 64C in the midgut, 256C in Malpighian tubules, and the modal values are 32C and 128C respectively. The data provide no evidence for extensive underreplication of heterochromatin. It is suggested that the reduced amount of satellite DNA found in the tissues of young adult flies may be a consequence of the fact that cycles of DNA replication started in the pre-adult stages are not completed until some hours after eclosion.     

Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro‐CT

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro‐computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a ‘glandular band’ by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio‐opaque concretions. Moreover, the anatomy of O. ovis third‐instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a ‘band’ and anterior Malpighian tubules narrowly uniform throughout their entire length.     

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Summary Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm^–3 in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2–30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).     

Chromosomal DNA variation in Cucumis

Summary Variation in nuclear DNA amounts found in different species of Cucumis was surveyed. The DNA amounts varied from 1.373 to 2.483 pg in diploids and from 2.846 to 3.886 pg in tetraploids. DNA amount was not correlated with chromosome number and periodicity. Tetraploids were found to have double the quantity of nuclear DNA of diploids. A positive linear relationship was established between the nuclear DNA amounts and volume of chromosomes. The botanical varieties within a particular species do not differ significantly for 2C DNA amounts. A comparison of the distribution of DNA amounts among different chromosomes of haploid complement in different species revealed that the quantitative DNA changes associated with speciation affected all chromosomes. DNA changes were not however, of the same magnitude in all chromosomes of the complement. Speciation in Cucumis thus seems to have occurred through amplification or diminution of DNA proportionate to the size of chromosomes. The relationship between the basic numbers, x=7 and x=12, will have to be considered relative to the high DNA amount noticed in some species with x=12.     

Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito,Culiseta inornata (Williston)

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.     

Evolution of genome size and DNA base composition in reptiles

The evolution of genome size and base composition of DNA from various reptiles has been studied. DNA amount was measured cytophotometrically and GC concentration estimated by thermal denaturation. The Reptilia appear to be a fairly homogeneous group with respect to DNA quantity, although chelonians stand out because of their higher inter- and intrafamilial variability and DNA content. Quantitative DNA variations do not show a single evolutionary trend, but rather seem to have followed different patterns within each group.The differences in genome size between related species seem to be mainly the result of duplication or loss of DNA sequences characterized by a similar mean denaturation temperature. This agrees with observations of other authors that quantitative variations in reptiles are mainly due to differences in the amount of repetitive DNA.Several hypotheses on the significance of quantitative DNA variations in reptiles are discussed.     

DNA replication in binucleate cells of the Malpighian tubules of Hemipteran insects

The cells of Malpighian tubules of the Hemipteran blood-sucking insect, Rhodnius prolixus, are binucleate. The cells grow without division and, at each larval moult, the DNA content of the nuclei doubles. At the final moult to the adult, however, the DNA content does not change, even though the tubules grow considerably thereafter. In contrast, the DNA content of the tubule nuclei of two other Heteropteran Hemipteran insects, Dysdercus and Oncopeltus, doubles at every moult, including the final one to the adult. If extra larval moults are induced in Rhodnius, by treatment with juvenile hormone, DNA doubling is induced at each such supernumerary larval moult. Shortly after the DNA content increases in Rhodnius tubules, the chromosomes can be seen in a condensed state; presumably, therefore, DNA replication is achieved by endomitosis. Both before this DNA doubling, and within a day after, multiple nucleoli are prominent and appear actively engaged in producing ribosomal precursors. In fed fourth stage Rhodnius, the DNA content of the tubule cell nuclei increases 5–6 days after the blood meal. Neither the rate of fluid secretion that can be induced by stimulation nor the rate of transport of p-aminohippuric acid show any change at the time of DNA replication nor in the remaining days before ecdysis to the fifth stage. Malpighian tubule cell growth without division is thus well adapted to providing, without interruption, for the excretory needs of the growing insect.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Geopora</Emphasis> (Pyronemataceae,Pezizales)

Species delimitation in the genus Geopora (Pyronemataceae) is complicated because of small number of differentiating characters, values of which tend to overlap among the species. Current classification relies mainly on size and shape of ascospores and fruit-bodies, position of the apothecia in the ground and length of excipular hairs. We measured ascospores in ca. 90 Geopora specimens. Sequences of internal transcribed spacer (ITS) rDNA gene were obtained to investigate phylogenetic relationships in the genus. Maximum parsimony (MP) and Bayesian analyses reveal that the well-supported clades detected often do not correspond to the species concepts based on morphological characters. Nine out of the ten lineages include specimens which were initially identified as belonging to different species. The dimensions of ascospores vary to great extent within the lineages. The size and shape of fruit-bodies, length of excipular hair and hymenium colour are mostly homogenous within each clade; however, these characters coincide to a great extent among the lineages and the latter can be assessed only from fresh fruit-bodies. Nuclear DNA content, and accordingly, ploidy level do not provide evidence for species distinction. Geopora arenicola, G. tenuis and G. sepulta were recognized as monophyletic species. Geopora foliacea and G. cervina could not be explicitly delimited and the G. cervina complex comprising three clades was introduced.     

Purine transport by Malpighian tubules of pteridine-deficient eye color mutants of Drosophila melanogaster

Uptakes of guanine into Malpighian tubules of wild-type Drosophila and the eye color mutants white (w), brown (bw), and pink-peach (p ^p) have been compared. Tubules for each of these mutants are unable to concentrate guanine intracellularly. The transport of xanthine and riboflavin is also deficient in w tubules. The transport of guanosine, adenine, hypoxanthine, and guanosine monophosphate is similar in wild-type and white Malpighian tubules. These data and other information about these mutants make it likely that these pteridine-deficient eye color mutants do not produce pigments because of the inability to transport a pteridine precursor. This view supports the hypothesis that mutants which lack both pteridine and ommochromes do so because precursors to both classes of pigments share a common transport system.This work was supported by Grant GM22366 from NIH.     

The replicative status of heterochromatic and euchromatic DNA in two somatic tissues of Dermestes maculatus (Dermestidae: Coleoptera)

Replication of DNA present in euchromatin and heterochromatin remains in step in most nuclei of the testis wall of Dermestes maculatus. However, in a minority class of testis wall nuclei heterochromatic DNA has undergone two or three fewer rounds of replication than DNA located in euchromatin. A similar situation exists in many nuclei of the Malpighian tubule though here there is also the possibility that the euchromatic and heterochromatic DNA components are themselves not completely replicated.     

A Flow Cytometric Analysis of the Nuclear 2C DNA Content in 17 Phaseolus Species (53 Genotypes)

The genus Phaseolus is characterized by a highly stable karyotype of 2n = 22. Despite this constancy, the size of the chromosomes varies, and crossing of species is possible only in a few cases. We determined the 2C nuclear DNA content of a number of Phaseolus species, cultivars and genotypes by flow cytometry, in order to realize the interspecific and intraspecific variation of the 2C value. The data range from 1.03 pg to 2.18 pg without any clear correlation to systematic relationships. The mean DNA values of wild and cultivated forms, as well as those of Andean and Mesoamerican genotypes, do not differ significantly. The variation is interpreted in terms of some nucleotypic adaptations. The data may be useful for molecular biological analyses, as well as for biotechnological and classical breeding programmes.     

Constraints upon the organisation and evolution of chromosomes in Allium

Summary In terms of chromosome morphology, karyotype organisation, taxonomy and genetic relationship as judged from chromosome pairing in the Fl hybrid, A. cepa and A.fistulosum are two closely related species. But large variation in nuclear DNA amounts has occurred during the evolution of the two species. A comparison of the molecular composition of DNA in the two species has confirmed that the excess DNA acquired during evolution was predominantly repetitive sequences (sequences which do not encode genetic information). However, its distribution within the chromosome complements was equal in all chromosomes irrespective of the differences in chromosome size. The even distribution of the excess DNA within complements suggests strong constraints underlying evolutionary changes in genome organisation. The nature of the constraints is discussed, and it is shown that such constraints can influence the direction of karyotype evolution during speciation.     

Quantification and characterization of nuclear genomes in commercial red seaweeds (Gracilariales) from the Philippines

Eight species of Gracilariaceae from the Philippines, representing the generaGracilaria, Gracilariopsis andHydropuntia, were investigated to quantify and characterize their nuclear genomes. DNA reassociation kinetics were used to determine nuclear genome organization and complexity in six of these species. Results indicate the presence of three second order components corresponding to fast, intermediate and slow fractions. Repetitive sequences varied from 13–74% and unique DNA ranged from 26–84%. Microspectrophotometry with the DNA-localizing fluorochrome DAPI was used to quantify nuclear DNA contents. Comparisons of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.38–0.43 pg/2 C genomes for seven of the species investigated. Preliminary analyses of agar content and quality confirm the economic potential ofGracilaria firma, Gracilaria sp. 2 from Sorsogon andGracilariopsis bailinae. Nuclear genome profiles developed from data for genome size, organization and complexity are compared with data for agar quantity and quality. Gel quality and quantity do not appear to be correlated with either large repetitive fraction DNA or a high degree of genome complexity.Author for correspondence     

Cytophotometric evidence of variation in genome size of desmognathine salamanders

Summary The amount of DNA per haploid genome, the C-value, is often directly correlated with nuclear and cell volume, but inversely correlated with cell replication rate. Also, rates of cellular growth sometimes appear to be correlated with organismal developmental rates and life history patterns. Among vertebrates, salamanders exhibit the greatest variation in genome size. In the present study we have examined interspecific and intraspecific variation in blood cell DNA levels in the genus Desmognathus, which shows greater variation in life history traits than any other salamander genus. Specimens of Desmognathus quadramaculatus, D. Monticola, D. ochrophaeus and D. wrighti were collected from nature at two localities in the southern Appalachian Mountains. Estimates of genome size in pg of DNA were obtained from blood smears by DNA-Feulgen cytophotometry, using erythrocyte nuclei of Xenopus laevis as an internal reference standard of 6.35 pg DNA per cell. C-values of Desmognathus are the smallest in the order Caudata. Although significant variation in DNA levels was found among the four species, the differences were small, and do not support previously proposed relationships between C-value and life-history variation.     

Some morphometric,anatomical and biochemical characteristics of fruits and seeds of Onobrychis spp. in Italy

ABSTRACT

The fruits, seed coats and seed storage proteins of Onobrychis arenaria, O. montana, O. viciifolia, O. alba, O. supina and O. caput-galli were studied to examine the variability between and within species. The morphometric characteristics of the fruit had a high intraspecific variability which often exceeded that between species. Only the fruit of O. caput-galli had values for length, width, thickness and number of thorns which were almost always higher than those of the other species. The anatomical characteristics of the seed coats were extremely variable. The highest values of seed coat thickness were recorded in the diploid species. The palisade-like layer (or Malpighian cells) in O. caput-galli differed in size and morphology from that of the other species. The electrophoretic analysis revealed that the number and position of storage proteins varied between and within species.     

Malpighian tubules of larval Aedes aegypti are hormonally stimulated by 5-hydroxytryptamine in response to increased salinity

Two environmental parameters, feeding status and salinity, are expected to affect water and ion balance of the aquatic larvae of Aedes aegypti. Evidence was obtained for regulation of Malpighian tubule fluid secretion rates in response to changes in each of these parameters. Exposure to increased salinity induces release into the hemolymph of material with diuretic effects on Malpighian tubules. Diuretic material is present in hemolymph of larvae raised in higher salinities, rapidly appears in the hemolymph of larvae following transfer from dilute water to higher salinity, and rapidly disappears from the hemolymph following transfer from higher salinity to dilute water. Feeding status affects diuretic properties of both hemolymph and Malpighian tubules. Feeding causes hemolymph to become diuretic relative to hemolymph from nonfeeding larvae. Malpighian tubules removed from feeding larvae have greater basal fluid secretion rates and also appear to have greater maximal fluid secretion capacity than do tubules removed from nonfeeding larvae. Larval hemolymph [5-HT] was found to increase fivefold in response to elevated salinity but was unaffected by feeding status. Methiothepin, a 5-HT receptor antagonist, inhibited stimulation of fluid secretion by 5-HT and blocked the diuretic effects of hemolymph from larvae exposed to higher salinity but was without effect on stimulation of fluid secretion by diuretic peptide. During the course of this investigation, a preliminary pharmacological characterization of the 5-HT receptor on Aedes Malpighian tubules, suggesting that this receptor may be pharmacologically distinct from other described insect 5-HT receptors, was obtained. Arch. Insect Biochem. Physiol. 34:123–141, 1997. © 1997 Wiley-Liss, Inc.     

DNA‐based approaches for evaluating historical demography in terrestrial vertebrates

Contemporary DNA sequences can provide information about the historical demography of a species. However, different molecular markers are informative under different circumstances. In particular, mitochondrial (mt)DNA is uniparentally inherited and haploid in most vertebrates and thus has a smaller effective population size than diploid, biparentally inherited nuclear (n)DNA. Here, we review the characteristics of mtDNA and nDNA in the context of historical demography. In particular, we address how their contrasting rates of evolution and sex‐biased dispersal can lead to different demographic inferences. We do so in the context of an extensive review of the vertebrate literature that describes the use of mtDNA and nDNA sequence data in demographic reconstruction. We discuss the effects of coalescence, effective population size, substitution rates, and sex‐biased dispersal on informative timeframes and expected patterns of genetic differentiation. We argue that mtDNA variationin species with male‐biased dispersal can imply deviations from neutrality that do not reflect actual population expansion or selection. By contrast, mtDNA can be more informative when coalescence has occurred within the recent past, which appears to be the case with many vertebrates. We also compare the application and interpretation of demographic and neutrality test statistics in historical demography studies. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 367–386.     

Distinguishing Anuran species by high‐resolution melting analysis of the COI barcode (COI‐HRM)

Taxonomic identification can be difficult when two or more species appear morphologically similar. DNA barcoding based on the sequence of the mitochondrial cytochrome c oxidase 1 gene (COI) is now widely used in identifying animal species. High‐resolution melting analysis (HRM) provides an alternative method for detecting sequence variations among amplicons without having to perform DNA sequencing. The purpose of this study was to determine whether HRM of the COI barcode can be used to distinguish animal species. Using anurans as a model, we found distinct COI melting profiles among three congeners of both Lithobates spp. and Hyla spp. Sequence variations within species shifted the melting temperature of one or more melting domains slightly but do not affect the distinctness of the melting profiles for each species. An NMDS ordination plot comparing melting peak profiles among eight Anuran species showed overlapping profiles for Lithobates sphenocephala and Gastrophryne carolinensis. The COI amplicon for both species contained two melting domains with melting temperatures that were similar between the two species. The two species belong to two different families, highlighting the fact that COI melting profiles do not reveal phylogenetic relationships but simply reflect DNA sequence differences among stretches of DNA within amplicons. This study suggests that high‐resolution melting analysis of COI barcodes (COI‐HRM) may be useful as a simple and rapid method to distinguish animal species that appear morphologically similar.