6th Pair Research Articles

Comparative cytogenetics of Hemorrhois hippocrepis and Malpolon monspessulanus highlights divergent karyotypes in Colubridae and Psammophiidae (Squamata: Serpentes)

Despite the growing interest in the evolutionary cytogenetics of squamates, chromosomal data are lacking for most taxa. We performed a preliminary molecular taxonomic analysis and a comparative cytogenetic study on Hemorrhois hippocrepis and Malpolon monspessulanus. We used a combination of standard karyotyping, Chromomycin A3/Methyl green staining, C-banding, Ag-NOR staining and NOR-FISH to provide the first karyotype description of H. hippocrepis and a re-description of the karyotype of M. monspessulanus, including chromosome markers, heterochromatin patterns and sex chromosome systems. Our results show that H. hippocrepis has 2 n = 36 chromosomes, with 16 macro- and 20 microchromosomes and NORs on the 6th pair. The 4th pair represents homomorphic (metacentric) ZW sex chromosomes, but the W chromosome is completely heterochromatic. Malpolon monspessulanus has 2 n = 44 chromosomes, with 20 macro- and 24 microchromosomes, NORs on the 6th telocentric pair. The 4th pair represents the sex chromosomes (ZZ/ZW), with a W chromosome smaller than the Z and completely heterochromatic. Comparing our cytogenetic data to those available from the literature, we note the occurrence and distribution of primitive and derived chromosomal characteristics and discuss the chromosome diversification in two snake clades belonging to Colubridae and Psammophiidae, respectively. We highlight that these two families followed different chromosome diversification pathways, characterised by a highly conserved karyotype structure in Colubridae and a higher chromosome variability in Psammophiidae, mostly driven by a progressive reduction of the chromosome number by means of chromosome fusions. We also provide cytotaxonomic insights supporting the distinction between M. monspessulanus and M. insignitus.

Two species of the genus Anchistrocheles (Bairdioidea: Ostracoda: Crustacea) from Japan and their developmental characteristics for adaptation to interstitial environments

A new species and an already known species of the genus Anchistrocheles Brady & Norman, 1889 from the Pacific coast of Japan, are described in detail using scanning electron microscopy of the carapaces and soft-part anatomy of the appendages. The new species, Anchistrocheles hayatotanakai sp. nov., is only the second to be described in the genus Anchistrocheles from Japan, and this study is also the first to describe the appendages of A. yamaguchii Yajima, 1987, previously only known as a fossil from Pleistocene strata of the Atsumi Formation in central Japan. The two species are considered to be closely related because they share many common characters in their appendages, but they are distinguishable from each other based on the male and female copulatory organs, furcal chaetotaxy, brush-shaped organs, number of lateral pore systems, and outlines of the carapaces in dorsal view. Ontogenetic changes of carapace sizes were compared between three interstitial species (Anchistrocheles hayatotanakai sp. nov., A. yamaguchii, and Neonesidea sp. I) and an epifaunal species (N. sp. S). The reduction in carapace width was found to be the primary reason for the reduction in the cross-sectional area and is probably related to the adaptation to interstitial environments. The brush-shaped organs located between the 6th pair of limbs are also shown. This study is the first to report the anatomically precise position of these organs.

Karyotype diversity in cultivated and wild Indian rice through EMA-based chromosome analysis

India is known for its diverse cultivated and wild rice germplasm. In today's crop improvement programmes, wild relatives are much-needed genetic repository of valuable traits. Analysis of genetic diversity at the chromosomal level is one cost-effective tool to unlock foundational information related to genetics and plant breeding. Presently, enzymatic maceration and air-drying method (EMA) has been applied for the first time in six cultivated and nine wild Indian rice (diploid and tetraploid). EMA method following Giemsa staining has yielded large numbers of cytoplasm free metaphase plates with distinct chromosome morphology. Detailed analysis has revealed karyotype diversities in terms of total chromatin length (TCL), chromosome morphology and location of sat chromosomes within and between the studied species. Most of the cultivated rice has gained additional amount in TCL during the period of domestication in comparison to their progenitor Oryza nivara. Morphological clarity of the small chromosomes of rice was much required and has helped to identify individual chromosomes in the diverse karyotypes. Diversity in landmark SAT chromosomes is another important observation, not reported previously in Indian rice. Present study has shown that in most of the O. sativa members, the 10th pair contains SAT except one where 6th pair is satellited. On the other hand, diversity of SAT in diploid and tetraplod wild species has been recorded on 5th, 7th and 8th chromosome pairs and on 9th, 12th, 22nd and 23rd chromosome pairs, respectively. Karyomorphometric indices has helped to construct dendrogram to elucidate intraspecies and interspecies relationships. Untapped genetic diversity recorded in Indian rice through chromosomal analysis will be useful to the breeders and genome researchers.

Comparative cytogenetic of six species of Amazonian Peacock bass (Cichla, Cichlinae): intrachromosomal variations and genetic introgression among sympatric species.

Cytogenetic data for the genus Cichla Bloch et Schneider, 1801 are still very limited, with only four karyotype descriptions to date. The sum of the available cytogenetic information for Cichla species, points to a maintenance of the diploid number of 48 acrocentric chromosomes, considered a typical ancestral feature in cichlids. In the current study, we performed molecular and classical cytogenetic analyses of the karyotype organization of six species of Cichla, the earliest-diverging genus of Neotropical cichlids. We cytogenetically analysed Cichla kelberi Kullander et Ferreira, 2006, Cichla monoculus Agassiz, 1831, Cichla piquiti Kullander et Ferreira, 2006, Cichla temensis Humboldt, 1821, Cichla vazzoleri Kullander et Ferreira, 2006 and Cichla pinima Kullander et Ferreira, 2006, including three individuals that showed mixed morphological characteristics, likely from different species, suggesting they were hybrid individuals. All individuals analysed showed 2n = 48 acrocentric chromosomes, with centromeric heterochromatic blocks on all chromosomes and a terminal heterochromatic region on the q arm of the 2nd pair. Mapping 18S rDNA gave hybridization signals, correlated with the nucleolus organizer regions, on the 2nd pair for all analyzed individuals. However, we found distinct patterns for 5S rDNA: interstitially at the proximal position on 6th pair of four species (C. kelberi, C. pinima, C. piquiti and C. vazzoleri), and on the distal of the 4th pair in two (C. monoculus and C. temensis). Accordingly, we present here new data for the genus and discuss the evolutionary trends in the karyotype of this group of fish. In addition, we provide data that supports the occurrence of hybrid individuals in the Uatumã River region, mainly based on 5S rDNA mapping.

Topotype-based redescription of the leech Torix tukubana (Hirudinida: Glossiphoniiformes: Glossiphoniidae)

A poorly-known proboscidate leech species, Torix tukubana (Oka, 1935), in which the mid-body somites are biannulate dorsally and triannulate ventrally, is redescribed based on new specimens collected from its type locality, Mt. Tsukubasan in Honshu, Japan. The redescription provides the internal digestive and genital organs of T. tukubana for the first time. Our observation reveals that this species possesses equal-sized 1st–6th pairs of crop ceca that are nondiverticulated and tubular ovisacs running alongside the ventral nerve cord. Additionally, phylogenetic analyses using nuclear 18S rRNA, mitochondrial cytochrome c oxidase subunit I, 12S rRNA, tRNALeu, and NADH dehydrogenase subunit 1 markers reveal that T. tukubana is closely related to the Palearctic Hemiclepsis Vejdovský, 1884, in which the mid-body somites are triannulate.

First report of bicolour FISH of Berberis diaphana and B. soulieana reveals interspecific differences and co-localization of (AGGGTTT)3 and rDNA 5S in B. diaphana

BackgroundBerberis consists of approximately 500 species and is the largest genus in Berberidaceae. Most Berberis species lack cytological data, and bicolour fluorescence in situ hybridization (FISH) has never been performed on Berberis. In this work, a karyotype of Berberis diaphana, an alpine Berberis species obtained from an altitude of 3600 m in Wolong National Nature Reserve, China, was analysed and compared with Berberis soulieana Schneid. via FISH using oligonucleotide telomere probes for (AGGGTTT)3 and 5S rDNA (41 bp) for the first time.ResultsBerberis diaphana belonged to cytotype 2A and had the karyotype formula 2n = 2x = 28 = 26 m + 2 sm (2SAT). The mitotic metaphase chromosome lengths ranged from 1.82 ± 0.04 μm to 2.75 ± 0.00 μm. Clear (AGGGTTT)3 signals were detected at two telomeres in every chromosome and were co–localized with 5S rDNA at the terminal regions of the long arms in the 6th pair of chromosomes. One pair of (AGGGTTT)3 sites was localized in the satellites of the 7th pair of chromosomes, which are the only submetacentric chromosomes in this species. Totally 28 chromosomes with one pair of satellited chromosomes were observed in B. soulieana. This species had four 5S rDNA signals with two weak signals at the end of long arms in the 5th pair of chromosomes and another two strong signals detected in the interstitial region close to the end of short arms in the 6th pair of chromosomes. Each large signal consisted of two smaller signals with secondary constrictions around them.ConclusionsFISH physical mapping of B. diaphana suggested that (AGGGTTT)3 and rDNA 5S co-localize at the 6th pair of chromosomes. The density, location and number difference of 5S rDNA loci indicated structural differences among the chromosomes between B. diaphana and B. soulieana. Our results provide information that may contribute to future studies on the physical assembly of the Berberis genome and the evolution of rDNA and telomere FISH patterns in Berberis.

Ecology of Vespidae (Hymenoptera)Predators in Coffea arabica Plantations

Social Vespidae exhibit control of Leucoptera coffeella (Lepidoptera: Lyonetiidae) in Brazil. The objective was to determine the ideal unit for sampling of predaceous Vespidae in coffee crops in the vegetative and reproductive phases. This research was conducted in two coffee plantations in Viçosa, MG. The factors being studied were: crop phase, canopy thirds, branch type, exhibition side of the plant to solar light and the position of the leaf on the branch. The number of predation mines by Vespidae on all the leaves of each evaluated plant was recorded. In coffee plants in the vegetative phase the best sampling unit of the Vespidae was the 5th or 6th pair of leaves on the primary plagiotropic branches of the median third of the canopy. In coffee plants already in the reproductive phase the best unit for sampling Vespidae were leaves on the third apical of the 4th or 6th pair of leaves on primary plagiotropic branches on the plant face exposed to the sun in the afternoon period or on the median third on the 5th pair of leaves of the plant face exposed to the sun in the afternoon period.

Preliminary cytogenetic study and report of ZZ/ZW sex chromosomes in the bullfrog, Hoplobatrachus tigerinus (Anura, Amphibia) from high altitude area of Jammu and Kashmir, India

Cytogenetic study of the Indian Bullfrog, Hoplobatrachus tigerinus was carried out using both conventional staining and banding methods, from Jammu and Kashmir. Karyotypes prepared by conventional staining in both male and female specimens showed diploid chromosome number as 2n = 26 and fundamental arm number as NF = 52. Karyotypes of both the sexes comprised of 13 pairs of biarmed chromosomes divided into two groups: first group of five pairs of larger metacentric or submetacentric chromosomes and second group of eight pairs of smaller metacentric or submetacentric chromosomes. Pair number 2, 3, and 4 among the first group and 8 and 9 among the second group were submetacentric chromosomes. All the other chromosomes in both the groups were metacentric. Meiotic stages observed were leptotene, diakinesis, metaphase-I and metaphase- II, from testicular cells of males. C-banding analysis of the species showed presence of centromeric C-bands in all the chromosomes of both male and female karyotypes. Female karyotype showed presence of completely heterochromatic homologue of the pair no. 8, which represented ZZ/ZW sex chromosome system in the species. NOR-banding showed presence of a pair of nucleolar organizer regions on the long arm of 6th pair in both the sexes.

Physical mapping of 18S rDNA cistron in species of the Omophoita genus (Coleoptera, Alticinae) using fluorescent in situ hybridization

Alticinae has the greatest amount of biodiversity among the Chrysomelidae, with 40,000 described species, only 290 of which have been analyzed cytogenetically. The majority of studies refer to conventional staining and few species have been analyzed or have responded to differential staining methods. The aim of the present study was to describe an 18S rDNA probe for Alticinae and the location of this cluster in species of the Omophoita genus. The fragment of approximately 750 bp obtained through a PCR (Polymerase Chain Reaction) amplification reaction with specific oligonucleotides to 18S rDNA was cloned and denominated pTZ_Ooct_18Sp and then submitted to automatic sequencing. The alignment of the sequences obtained through the sequencing of the clones generated a consensus sequence of 722 bp for Omophoita octoguttata with 98% homology with other species of Alticinae. The analysis of mitotic cells of O. octoguttata and Omophoita magniguttis submitted to fluorescent in situ hybridization (FISH) with the 18S rDNA probe revealed that the ribosomal genes are located in 6th pair. O. magniguttis also has a second labeled pair. Omophoita personata exhibited nucleolar organizer regions associated to one autosome pair. The analysis of meiotic cells submitted to FISH revealed one labeled bivalent in metaphase I in O. octoguttata and O. personata and in one chromosome in metaphase II in O. octoguttata. FISH data suggest a conserved pattern in the species analyzed and an apomorphy of O. magniguttis karyotype. The rDNA 18S probe could be considered an important marker to evidence the karyotypic differentiation, not observed with conventional methodologies, in species considered karyotypically conserved and uniform.

Derived cytogenetic traits, multiple NORs and B chromosomes in the compact karyotype of Canthigaster figueiredoi (Tetraodontiformes)

The Tetraodontiformes are one of the main irradiation lineages of Teleosts representing a highly specialized group. Among its families, Tetraodontidae has the lowest DNA/cell content of the vertebrates, and for this reason has been used as a model group for genomic evolution studies. Seeking to widen the cytogenetic database of this family, we performed a chromosomal analysis of the species Canthigaster figueiredoi using conventional staining, Ag-NORs, C-banding, base-specific fluorochromes DAPI-CMA 3, and in situ hybridization with 18 S rDNA probe. This species has 2n = 36 (10 m + 6sm + 20a; FN = 52). Multiple Ag-NORs (CMA 3 +) sites were detected on the four chromosome pairs. Heterochromatic blocks (CMA 3 +) were present in the centromeric regions of most of the pairs, extending on the short arm of some chromosomes. A GC-positive polymorphic heterochromatic region was identified in some of the individuals, in one or both of the homologs of the 6th pair. The presence of heteromorphic B microchromosomes was detected in the karyotype of one female, exhibiting intra-individual variation of 0–3 Bs. The occurrence of heterochromatic polymorphisms, multiple NORs, and B chromosomes in C. figueiredoi are very infrequent events in marine fish. They are probably associated to the accentuated restructuring and genomic reduction suffered by this family.

Evidence for a remarkable stasis of chromosome evolution in Malagasy treefrogs (Boophis, Mantellidae)

A comparison of chromosome morphology of seventeen species of Boophis (Mantellidae, Boophinae) was carried out applying Giemsa, C‐, DAPI‐, CMA3and AgNOR‐ banding methods. All of the species studied showed a karyotype of 2n = 26 biarmed chromosomes. The nucleolar organizer regions (NORs) were interstitially located in a secondary constriction on the long arm of the 6th chromosome pair in all species, except B. cf. rufioculis and B. cf. madagascariensis in which the NORs were dislocated on the short arms of the 6th pair. Heterochromatin distribution and composition was very variable among the species, displaying C‐bands positive or negative both to DAPI and CMAj, or positive only at one of the two fluorochromes. Taxonomic and phylogenetic implications of these results are discussed.

Megacystis‐microcolon‐intestinal hypoperistalsis syndrome (MMIHS), an autosomal recessive disorder: Clinical reports and review of the literature

We present 2 new patients with the megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS), review the literature, and discuss the prenatal diagnosis and treatment. MMIHS, as reported in 43 cases, is usually lethal. Most children die during the first year of life, and only 3 children survived their first year. We report the 6th pair of sibs with the disease. Overall, 17 patients reported have had sibs with MMIHS or the parents were consanguineous; 4 times the parents were first, cousins, confirming that this is an autosomal recessive disorder. The present 2 children, whose parents also were first cousins, were of different sex. They had typical MMIHS with abdominal distension due to pronounced megacystis, hydronephrosis, microcolon, and microileum, involving the distal part of the ileum, malrotation of the gut, and intestinal hypoperistalsis. Neither surgery nor medical treatment was successful and the children died at the age of 19 days and 2 1/2 months, respectively. There is no cure for the disease. However, a new protkinetic drug, Cisapride might be worth trying in these cases. Prenatal ultrasound diagnosis of MMIHS might be possible.

The transition to reproductive phase inchenopodium murale L. ecotype 197 - Early flowering long-day plant

Five days of suitable continuous light induced flowering in the majority ofChenopodium murale L. ecotype 197 plants as early as at the phase of the first pair of leaves. At the time of initiation of the 2nd to 4th pairs of leaves the capacity of plants to flower was reduced, the number of flowering plants being significantly lower under the same inductive light treatment. The capacity to flower increased again at the phase of the 5th and the 6th pairs of leaves. Inductive light treatment brought about a marked growth activation of organs present before induction, shoot apex elongation, precocious formation of new leaves and activation of axillary meristems. The course of these changes in plants of different age is demonstrated. The terminal flower developed during 5 short days following inductive light treatment. The paper shows similarities and differences between long-daymutale L. ecotype 197 and short-day C.rubrum L. ecotype 374 grown under practically uniform conditions.

Chromosomal polymorphism of Bufo bufo: Karyotype and C-banding pattern of B. b. verrucosissima

Bufo bufo verrucosissima has a karyotype consisting of 22 chromosomes (6 pairs of large and 5 pairs of small chromosomes which are meta- and submetacentric). By means of Ag-AS-staining nucleolar organizers were localized in the telomeric region of the long arms of the 6th pair of chromosomes. The karyotype differs from those of the other B. bufo subspecies by the form of the 4th pair, which is metacentric. A slight chromosomal polymorphism was shown also after C-banding of B. b. verrucosissima and B. b. bufo chromosomes.

Familial Urinary Tract Anomalies: Association with the Major Histocompatibility Complex in Man

Histocompatibility typing of a family with 15 members and a history of ureteropelvic junction stenosis and 4 families with 23 members and a history of vesicoureteral reflux revealed that these anomalies of the urinary tract may be hereditary and segregate with histocompatibility haplotype within a family. Thus, a close linkage of childhood reflux and ureteral stenosis with that of the major histocompatibility complex of man is suggested. If confirmed by further family studies it will place the gene(s) for vesicoureteral reflux and ureteral stenosis on the 6th pair of human chromosome and open the possibility of using histocompatibility typing as a marker for these anomalies within a family

Karyotype and heterochromatin pattern of the field mouse,Apodemus argenteus Temminck

The field mouse,Apodemus argenteus Temminck, has 46 chromosomes. The autosomes comprise 20 pairs of acrocentrics and 2 pairs of metacentrics. The X chromosome is represented by an outstandingly large submetacentric element, while the Y is an acrocentric corresponding in size to the 5th or 6th pair of autosomes. All of the autosomes and gonosomes can be unequivocally identified by their characteristic Q-band or G-band patterns. The constitutive heterochromatin, as revealed by C-banding, is localized at the centromeric regions of all autosomes, the short arm and the proximal 1/3 of the long arm of the X chromosome, and the entire Y chromosome. The C-band-positive segments which constitute 33.5% of the genome exhibit dark fluorescence after Q-banding, late DNA replication, faint or positive staining reaction to G-banding, fast reassociation of DNA revealed by AO staining, and allocyclic behavior of the sex-bivalent in male meiosis. An exception to the above is the distal segment of the Y which is positive to both C- and Q-banding. The giant X chromosome occupies 13.1% of the genome, leaving 5.6% of euchromatic segments, the latter value being equivalent to that of the original type X.

Aortic arch anomalies associated with the administration of epinephrine in chick embryos.

AbstractSingle doses of epinephrine (5 μg) administered to 83 groups of chick embryos (24–190 h) at 2‐h intervals, produced a spectrum of aortic arch anomalies. The epinephrine was placed on the surface, of the extra embryonic membrane over the developing embryo in ovo, at a time when aortic arch morphogenesis is occurring. The peak incidence of anomalies (94%) occurred after treatment during day 5 of incubation (108–112 h). The anomalies involved the 3rd, 4th, and 6th pairs of aortic arches; those involving the right 4th arch (arcus aortae) were consistently associated with ventricular septal defects.