Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae).

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae).

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Imitate to integrate: reviewing the pathway for B chromosome integration in Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae).

B chromosomes are genomic "intruders" normally characterized by their total dispensability counteracted by a variety of drive mechanisms, which assures their presence regardless of their harmful effects on the host genome. From an evolutionary standpoint, the relationship between standard (A) and B chromosomes can go through different pathways, from an everlasting arms race to a cordial B integration. Examples underlying the first situation are fairly common; B integration, however, has been more a theoretical than a practical possibility. The B chromosome in the haplodiploid solitary wasp Trypoxylon albitarse is probably the first example of a "mimetic" B, which is being integrated into the A genome by limiting itself to one B per haploid genome, the same dosage as the A chromosomes. Here we review some of the findings underlying this hypothesis and discuss the T. albitarse B strategy as a possible mechanism for B chromosome integration as a regular member of the chromosome complement in haplodiploid organisms.

Dinoponera lucida Emery (Formicidae: Ponerinae): the highest number of chromosomes known in Hymenoptera.

We report the remarkable karyotype of Dinoponera lucida, a Brazilian endemic ponerine ant. Its chromosome number is 2n=106, most of the chromosomes are acrocentric and of very small size, and the karyotype formula is 88A+18M. A chromosome pair of the AM(t) type is reported. This is the largest number of chromosomes reported for the Hymenoptera order until now.

Longitudinal differentiation in Melipona mandacaia (Hymenoptera, Meliponini) chromosomes.

Melipona mandacaia is a stingless bee endemic to northeast Brasil. We describe the M. mandacaia karyotype using C-banding technique. fluorochrome staining and treatment with restriction enzymes and discuss the position of this species in the context of the phylogeny of the genus. Melipona mandacaia has 2n = 18 (14 SM + 2 M + 2 A). Heterochromatin was detected in the pericentromeric region of pairs 1, 2 and 8 and in the form of small blocks in the remaining pairs. Staining with base-specific fluorochromes showed that this heterochromatin was rich AT (QM and DAPI), except in the region corresponding to the NOR which was rich GC (CMA3) and was cleaved by the HaeIII enzyme. Melipona mandacaia is a member of Group I Melipona. Treatment with DraI/Giemsa discloses a larger number of bands than treatment with DraI/QM. Pre-cleavage with DraI gave rise to a larger number of bands following QM staining; a circumstance evidently due to a removal of the DNA-protein complex that prevented the association of the fluorochrome with AT-rich DNA. The results highlight the complex nature of heterochromatin.

Genetic load caused by variation in the amount of rDNA in a wasp.

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount.

Integration of a B chromosome into the A genome of a wasp, revisited.

A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.

Integration of a B chromosome into the A genome of a wasp.

B chromosomes are genome symbionts, the presence of which in many eukaryote species is explained, in most cases, by their violation of Mendelian rules, usually based on meiotic or mitotic instability, leading to their accumulation in the germ line (drive). However, B chromosome integration into the genome as a regular member of the chromosome set should imply the loss of drive. A possible way of bypassing this difficulty is to regularize meiosis when the B chromosome is frequent in the population, in order to yield gametes with one B chromosome. In diploid organisms, this task needs to be achieved in the two sexes, but in haplodiploids the problem simplifies to only the diploid sex. We have found, to the authors' knowledge, the first evidence of a B chromosome that is regularizing its meiotic behaviour and limiting its number to one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, in the solitary wasp Trypoxylon albitarse. It suggests a possible mechanism for B chromosome integration as a regular member of the chromosome complement.

Silver staining of nucleolar organizer regions (NOR) in some species of Hymenoptera (bees and parasitic wasp) and Coleoptera (lady-beetle).

Adaptations of the nucleolar organizer regions (NOR) banding technique using precipitation of silver salts significantly improved the NOR characterization of some species of hymenopterans and one coleopteran. The bee Melipona marginata (2n = 18) showed one metacentric pair of chromosomes with a NOR in the pericentromeric position. The parasitic wasp Mellitobia australica (2n = 12) also showed one metacentric pair with a strongly Ag-positive NOR. The male lady-beetle Cycloneda sanguinea (2n = 18 + Xy(p)) displayed a NOR on a pair of acrocentric autosomes. In the male Euglossa sp. (a haplodiploid species) (n = 21) the NOR were multiple, and occurred in five chromosomes. In the bee Plebeia sp. 1 (2n = 34) the NOR seemed restricted to one of the homologues of a metacentric pair. The systematic advances brought out by using this technique in the context of current theories of karyotypic evolution of these taxa are described and discussed.

Sequential FISH analysis with rDNA genes and Ag-NOR banding in the lady beetle Olla v-nigrum (Coleoptera: Coccinellidae).

We have characterized the meiosis of Olla v-nigrum by standard analysis, performed a NOR study using NOR banding, FISH of rDNA genes and sequential FISH/AgNOR analysis, and adapted the FISH methodology to Coccinellidae. The chromosome number determined at metaphase I was n = 9 + Xyp. At zygotene it was possible to identify the sex vesicle which presented a deeply stained heteropycnotic block. Chromosome X is much larger than the y and the two combine, forming a "parachute" in metaphase I. FISH analysis using a probe of rDNA genes 18S, 28S and 5.8S of D. melanogaster was used to map the genes in the sex vesicle. The NOR band showed high gene activity in this region. These results were confirmed using sequential FISH/Ag NOR analysis. The data obtained for Olla v-nigrum agree with the classical hypothesis raised to explain the type of sex chromosome association in a parachute format (Xyp) as being due to the presence of nucleolar material. The chromosome number and parachute configuration during metaphase I in this species agree with the basic karyotype of most Coleopterans. The major adaptation of the FISH method was the simultaneous denaturation and hybridization that permitted preservation of chromosome morphology, an essential factor when the chromosomes are small.

The B chromosome system of Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae) 1. Banding analysis.

Karyotypic analyses of 366 specimens of the solitary wasp Trypoxylon (Trypargilum) albitarse collected from ten populations in the municipalities of Viçosa and Porto Firme (Minas Gerais, Southeastern Brazil), revealed the presence of two morphological types of supernumerary (B) chromosomes. C-banding and fluorochrome banding suggest that the B chromosomes of T. albitarse may have originated from heterochromatin breaks within the standard (A) chromosome complement.