Six new species of the Anopheles leucosphyrus group, reinterpretation of An. elegans and vector implications

Abstract. Among Oriental anopheline mosquitoes (Diptera: Culicidae), several major vectors of forest malaria belong to the group of Anopheles (Cellia) leucosphyrus Dönitz. We have morphologically examined representative material (> 8000 specimens from seven countries) for taxonomic revision of the Leucosphyrus Group. Six new species are here described from adult, pupal and larval stages (with illustrations of immature stages) and formally named as follows: An. latens n. sp. (= An. leucosphyrus species A of Baimai et al., 1988b), An. cracens n. sp., An. scanloni n. sp., An. baimaii n. sp. (formerly An. dirus species B, C, D, respectively), An. mirans n. sp. and An. recens n. sp. Additionally, An. elegans (James) is redescribed and placed in the complex of An. dirus Peyton & Harrison (comprising An. baimaii, An. cracens, An. dirus, An. elegans, An. nemophilous Peyton & Ramalingam, An. scanloni and An. takasagoensis Morishita) of the Leucosphyrus Subgroup, together with An. baisasi Colless and the An. leucosphyrus complex (comprising An. balabacensis Baisas, An. introlatus Baisas, An. latens and An. leucosphyrus). Hence, the former Elegans Subgroup is renamed the Hackeri Subgroup (comprising An. hackeri Edwards, An. pujutensis Colless, An. recens and An. sulawesi Waktoedi). Distribution data and bionomics of the newly defined species are given, based on new material and published records, with discussion of morphological characters for species distinction and implications for ecology and vector roles of such species. Now these and other members of the Leucosphyrus Group are identifiable, it should be possible to clarify the medical importance and distribution of each species. Those already regarded as vectors of human malaria are: An. baimaii[Bangladesh, China (Yunnan), India (Andamans, Assam, Meghalaya, West Bengal), Myanmar, Thailand]; An. latens[Borneo (where it also transmits Bancroftian filariasis), peninsular Malaysia, Thailand]; probably An. cracens (Sumatra, peninsular Malaysia, Thailand); presumably An. scanloni (Thailand); perhaps An. elegans (the Western Ghat form of An. dirus, restricted to peninsular India); but apparently not An. recens (Sumatra) nor An. mirans[Sri Lanka and south-west India (Karnataka, Kerala, Tamil Nadu)], which is a natural vector of simian malarias. Together with typical An. balabacensis, An. dirus and An. leucosphyrus, therefore, the Leucosphyrus Group includes about seven important vectors of forest malaria, plus at least a dozen species of no known medical importance, with differential specific distributions collectively spanning > 5000 km from India to the Philippines.     

Meiotic differences between three triatomine species (Hemiptera,Reduviidae)

We have found the following differences in the male meiosis among three triatomine species: (1) The three largest autosomal bivalents ofTriatoma infestans are heterochromatic.Rhodnius prolixus has two autosomal bivalents with heterochromatic blocks.Triatoma rubrovaria does not show any heteropycnotic autosomes. (2) Sex chromosomes inT. infestans form a chromocenter. At early prophase terminal associations are seen between sex chromosomes inT. rubrovaria, and they maintain a close association until diakinesis. An intimate association between the X and Y chromosomes is observed during early prophase inR. prolixus, but a distant association is maintained by the sex chromosomes at diffuse and diplotene stages in this species. (3) Polyploid nuclei of the nutritive cells are quite distinct. Numerous chromocenters of different shapes and sized are seen in those ofT. infestans. InT. rubrovaria one chromocenter having two positively heteropycnotic elements is observed surrounded by homogeneous chromatin. Only one compact chromocenter is found amongst unevenly distributed chromatin, inR. prolixus.     

Comparative cytogenetics of Diphyllobothrium ditremum (Creplin, 1925) and Ligula intestinalis (Linnaeus, 1758) (Cestoda: Pseudophyllidea)

An investigation carried out on two species of pseudophyllidean cestodes belonging to different families showed very close karyological affinity between them. The karyotypes of Diphyllobothrium ditremum and Ligula intestinalis both consist of 18 bi-armed chromosomes and are almost identical with respect to the relative length and the centromeric indices of corresponding chromosomes. Statistically significant differences exist in the morphology of chromosomes 2 and 4, but they are not striking and may be due in part to errors of measurement. Differences in the absolute length of the chromosomes were noted: the chromosomes of D. ditremum are somewhat larger (from 2.7 to 8.5 μm) than those of L. intestinalis (from 1.9 to 5.4 μm). The results obtained were compared with data existing for other pseudophyllidean cestodes and preliminary conclusions on the karyotypic evolution in that group of helminths were made.     

Sexual difference in juvenile-hormone titer in workers leads to sex-biased soldier differentiation in termites

In termites, the soldier caste, with its specialized defensive morphology, is one of the most important characteristics for sociality. Most of the basal termite species have both male and female soldiers, and the soldier sex ratio is almost equal or only slightly biased. However, in the apical lineages (especially family Termitidae), there are many species that have soldiers with strongly biased sex ratio. Generally in termites, since high juvenile hormone (JH) titer is required for soldier differentiation from a worker via a presoldier stage, it was hypothesized that the biased soldier-sex ratio was caused by differences in JH sensitivity and/or JH titer between male and female workers. Therefore, we focused on the presoldier differentiation and the worker JH titer in species with only male soldiers (Nasutitermes takasagoensis) and with both male and female soldiers (Reticulitermes speratus) in natural conditions. In the former species, there are four types of workers; male minor, male medium, female medium and female major workers, and presoldiers differentiate from male minor workers. First, we tried to artificially induce presoldiers from male and female workers. In N. takasagoensis, the presoldier differentiation rate and mortality was significantly higher in male minor workers. Morphological analyses showed that both male and female induced presoldiers possessed normal soldier-specific morphologies. It was suggested that female workers, from which soldiers do not differentiate under natural conditions, also maintained the physiological and developmental potential for soldier differentiation. In R. speratus, however, no differences were observed in solder differentiation rate and mortality between male and female workers. Second, the JH titers of each sex/type of workers were quantified by high performance liquid chromatography–mass spectrometry in two different seasons (April and December). The results showed that, in N. takasagoensis, JH titer in male minor workers was consistently higher than those in other worker types. In R. speratus, in contrast, there were no significant differences in JH titers between male and female workers. These results suggested that, in N. takasagoensis, male minor workers maintain JH titers at a high level throughout a year, and this may cause the male-biased presoldier differentiation.     

Polymorphic chromosomal inversions in Anopheles moucheti,a major malaria vector in Central Africa

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.     

Parallel evolution of termite‐egg mimicry by sclerotium‐forming fungi in distant termite groups

Among the great diversity of insect–fungus associations, fungal mimicry of termite eggs is a particularly fascinating consequence of evolution. Along with their eggs, Reticulitermes termites often harbour sclerotia of the fungus Fibularhizoctonia sp., called ‘termite balls’, giving the fungus competitor‐free habitat within termite nests. The fungus has evolved sophisticated morphological and chemical camouflage to mimic termite eggs. To date, this striking insect–fungus association has been found in eight temperate termite species, but is restricted to the lower termite genera Reticulitermes and Coptotermes. Here, we report the discovery of a novel type of termite ball (‘Z‐type’) in the subtropical termite, Nasutitermes takasagoensis. Phylogenetic analysis indicated that the Z‐type termite ball is an undescribed Trechisporoid fungus, Trechispora sp., that is phylogenetically distant from Fibularhizoctonia, indicating two independent origins of termite‐egg mimicry in sclerotium‐forming fungi. Egg protection bioassays using dummy eggs revealed that Reticulitermes speratus and N. takasagoensis differ in egg‐size preference. A comparative study of termite ball size and egg‐size preference of host termites showed that both fungi evolved a termite ball size that optimized the acceptance of termite balls as a unit investment. Termite‐egg mimicry by these fungi offers a model case of parallel evolution. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 531–537.     

The diminution of heterochromatic chromosomal segments in Cyclops (Crustacea,Copepoda)

The chromosomes of Cyclops divulsus, C. furcifer, and C. strenuus, like those of several other Copepods, undergo a striking diminution of chromatin early in embryogenesis. The process is restricted to the presumptive soma cells and occurs at the 5th cleavage in C. divulsus, at the 6th and 7th in C. furcifer, and at the 4th in C. strenuus. The eliminated chromatin derives from the excision of heterochromatic chromosome segments (H-segments). Their chromosomal location is different in the three investigated species: Whereas in C. divulsus and C. furcifer the H-segments form large blocks — exclusively terminal in the former and terminal as well as kinetochoric in the latter — the germ line heterochromatin in C. strenuus is scattered all along the chromosomes. Extensive polymorphism exists with respect to the length of the terminal H-segments in C. furcifer, and with respect to the overall content of heterochromatin in the chromosomes of C. strenuus. In a local race of C. strenuus an extreme form of dimorphism has been found which is sex limited: females as a rule are heterozygous for an entire set of large (heterochromatin-rich), and a second set of small chromosomes in their germ line. Males are homozygous for the large set. In the first three cleavage divisions the H-polymorphism is solely expressed through differences of chromosome length. Following diminution the differences between homologous have disappeared. Feulgen cytophotometry demonstrates that in the three species the 1C DNA value for the germ line, as measured in sperm, is about twice that measured in somatic mitoses (germ line/soma C-values in picograms of DNA: C. strenuus 2.2/0.9, C. furcifer 2.9/1.44, C. divulsus 3.1/1.8). — The data imply that chromatin diminution is based on a mechanism which allows specific DNA segments, regardless of their location and size, to be cut out from the chromosomes without affecting the structural continuity of the remaining DNA. This mechanism may be analogous to that of prokaryotic DNA excision.     

Cytogenetic studies on <Emphasis Type="Italic">Metasequoia glyptostroboides</Emphasis>, a living fossil species

The chromosome morphology and meiotic pairing behavior in the pollen mother cells (PMCs) of Metasequoia glyptostroboides were investigated. The results showed that: (1) The chromosome number of the PMCs was 2n=22. (2) The PMCs developed in the successive manner, and the nucleoids in the dynamic development were similar to those of the other gymnosperms. (3) At prophase, most of the chromosomes were unable to be identified distinctively because the chromosomes were long and tangled together. The chromosome segments were paired non-synchronously. At pachytene, the interstitial or terminal regions of some bivalents did not form synapsis and the paired chromosomes showed difference in sizes, indicating that there were structure differences between the homologous chromosomes. (4) At diakinesis, the ring bivalents showed complicated configurations due to the differences in location and number of chiasmata. In addition, there were cross-linked bivalents. (5) At metaphase I, the chromosome configuration of each cell was 8.2II ⁰ + 1.1II + 1.3II ⁺ + 0.8I. Most of the chromosomes were ring bivalents, but some were cross-linked bivalents, rod bivalents, or univalents. (6) 15\% PMCs at anaphase I and 22\% PMCs at anaphase II presented chromosome bridges, chromosome fragments, micronuclei, and lagging chromosomes. Twenty seven percent microspores finally moved into one to three micronuclei. Twenty five percent pollens were abortive. The results indicated that the observed individual of M. glyptostroboideswas probably a parpcentric inversion heterozygote, and there were structural and behavioral differences between the homologous chromosomes. The chromosomal aberration of M. glyptostroboidesmay play an important role in the evolution of this relict species, which is known as a living fossil. Further evidence is needed to test whether the differences between homologous chromosomes were due to hybridization.     

Distribution of heterochromatin on the mitotic chromosomes of Musca domestica L. in relation to the activity of male-determining factors

In the housefly, male sex is determined by a dominant factor, M, located either on the Y, on the X, or on any of the five autosomes. M factors on autosome I and on fragments of the Y chromosome show incomplete expressivity, whereas M factors on the other autosomes are fully expressive. To test whether these differences might be caused by heterochromatin-dependent position effects, we studied the distribution of heterochromatin on the mitotic chromosomes by C-banding and by fluorescence in situ hybridization of DNA fragments amplified from microdissected mitotic chromosomes. Our results show a correlation between the chromosomal position of M and the strength of its male-determining activity: weakly masculinizing M factors are exclusively located on chromosomes with extensive heterochromatic regions, i.e., on autosome I and on the Y chromosome. The Y is known to contain at least two copies of the M factor, which ensures a strong masculinizing effect despite the heterochromatic environment. The heterochromatic regions of the sex chromosomes consist of repetitive sequences that are unique to the X and the Y, whereas their euchromatic parts contain sequences that are ubiquitously found in the euchromatin of all chromosomes of the complement. Received: 20 February 1998; in revised form: 11 May 1998 / Accepted: 23 May 1998     

Interstrain heterochromatin polymorphisms in Drosophila melanogaster

Neuroblast chromosomes of 16 Drosophila melanogaster laboratory stocks (15 wild type and 1 carrying the mutant vermilion) were carefully analyzed for Q-banding patterns and morphological characteristics, in all the mitotic phases. Two forms of intraspecific heterochromatin variations, involving three types of chromosomes, are described: 1) differences in the fluorescence pattern with regard to the Y chromosome and the centromeric heterochromatin of the pair II; 2) differences in the size of the heterochromatic segment of the X chromosome. An unambigous evidence of such variants was obtained by comparing homologous chromosomes in the F1 hybrids, as well as in the F2 offspring, where differences in appearance of the heteromorphic chromosomes was readily identified as to the parental origin. The possible evolutionary significance and the usefulness of such cytologically detectable genetic differences between various strains, are considered.This paper is dedicated to Prof. Claudio Barigozzi with gratitude for his guidance and the long collaboration     

Comparative karyotype studies between Spanish and French populations of Eliomys quercinus L.

The cytogenetic study of two populations of Eliomys quercinus L. (Rodentia, Gliridae), whose geographic ranges are relatively near, showed that numerical and morphological differences exist between their karyotypes. The French dormouse has a diploid number 2n=50, while the Spanish one had 2n=48. The sex chromosomes and the number of autosomal arms are identical in both populations. The morphological differences are limited to the autosomal metacentric pair 12 of the Spanish dormouse, which does not appear in the French dormouse. However, the latter possesses two pairs of acrocentric chromosomes (20, 24) which are absent in the Spanish dormouse. The bands that correspond to the q and p arms of pair 12 in the Spanish dormouse are identical to pairs 20 and 24 in the French one, respectively. Consequently, we consider the Spanish Eliomys quercinus and the French Eliomys quercinus as having a common ancestor. The Spanish form has originated by means of a Robertsonian translocation, Rb (20, 24), between pairs 20 and 24 of the common ancestor.     

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.     

Cytotaxonomical studies of West Bengal charophyta: Karyotype analysis in Chara braunii

The paper deals with cytotaxonomic studies of three forms ofChara braunii-f.coromandelina (n = 14), f.schweinitzii (n = 14) and f.kurzii (n = 28) from West Bengal (India). An improved technique involving the use of a pretreating agent permitted a detailed analysis of the karyotypes of the three taxa studied. It has been concluded that structural alterations of chromosomes and polyploidy are associated with evolution of phenotypic differences in theChara braunii complex. The phylogenetic status ofChara braunii in the light of the present investigation is discussed.     

New Y chromosomes and early stages of sex chromosome differentiation: sex determination in <Emphasis Type="Italic">Megaselia</Emphasis>

The phorid fly Megaselia scalaris is a laboratory model for the turnover and early differentiation of sex chromosomes. Isolates from the field have an XY sex-determining mechanism with chromosome pair 2 acting as X and Y chromosomes. The sex chromosomes are homomorphic but display early signs of sex chromosome differentiation: a low level of molecular differences between X and Y. The male-determining function (M), maps to the distal part of the Y chromosome’s short arm. In laboratory cultures, new Y chromosomes with no signs of a molecular differentiation arise at a low rate, probably by transposition of M to these chromosomes. Downstream of the primary signal, the homologue of the Drosophila doublesex (dsx) is part of the sex-determining pathway while Sex-lethal (Sxl), though structurally conserved, is not.     

A species specific satellite DNA family of Drosophila subsilvestris appearing predominantly in B chromosomes

This paper describes a species specific satellite DNA family (pSsP216) of Drosophila subsilvestris, a palearctic species of the D. obscura group. The pSsP216 family consists of tandemly arranged 216 bp repetitive units that are predominantly localized on B chromosomes. These chromosomes appear in variable numbers in the karyotype of this species. Some pSsP216 repeats can also be detected in the centromeric heterochromatin of the acrocentric A chromosomes. Two strains, one with and the other without B chromosomes, were investigated for sequence variability and for the location of this satellite DNA on the chromosomes. Among 16 clones of the 216 bp basic repeat unit an overall similarity of about 93% and no strain specific differences were found, indicating that the B chromosomes may have derived from the A chromosomes (probably the dots) by spontaneous amplification of the pSsP216 satellite DNA family.     

Marker chromosomes commonly observed in the genus Glycine

Summary Soybean [Glycine max (L.) Merr.] chromosomes were analyzed using the chromosome image analyzing system, CHIAS, and seven groups, including subgroups, were identified based on morphological characteristics. Two pairs of chromosomes were conspicuous in their morphological traits. One pair of chromosomes, which had the largest arm ratio among all the chromosomes, was commonly observed in the species in all three subgenera of the genus Glycine. These chromosomes also displayed a unique pattern after N-banding and were detected as marker chromosomes. G. soja, which is considered to be the ancestor of G. max, has two types of marker chromosomes. The lines that carry the same type as G. max may be the ancestors of G. max among the lines of G. soja. The morphological differences of the marker chromosomes within the species in the subgenus Soja are discussed in relation to the domestication process of soybean.     

Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization

Summary The chromosomes of the tetraploid wheats Triticum timopheevi (Genome AAGG) and T. araraticum (Genome AAGG) were C-banded at mitosis. The identity of the banded and unbanded chromosomes was then established by firstly making comparisons with the hexaploid species T. zhukovskyi which has the genome formula AAAAGG. Secondly, the meiotic pairing in F₁ hybrids between T. timopheevi and diploid wheats was examined by means of C-banding. The results showed that the banded chromosomes belonged to the G genome, while the unbanded chromosomes belonged to the A genome. Only one of the two pairs of satellited chromosomes had strong heterochromatic bands. The relationship between the genomes of T. timopheevi and T. dicoccum (Genome AABB) was then assessed at meiosis in hybrids between these species, using the techniques of C-banding and in situ hybridisation of a cloned ribosomal RNA gene probe. It was concluded that there were differences both in the amount and distribution of heterochromatin and also translocation differences between the species.     

Pairing and segregation of the sex chromosomes in X1X2-males of Dysdercus intermedius with a note on the kinetic organization of Heteropteran chromosomes

The existence of an X₁X₂-mode of sex determination is confirmed by a study of all meiotic stages in the male cotton stainer (X₁X₂ and pertinent stages in the female (X₁X₁ X₂X₂). In the male, the X-chromosomes are heterochromatic and pair end-to-end in early meiotic prophase. At diakinesis, they disjoin and align side-by-side in the center of the spindle, forming a pseudotetrad. Anaphase I is equational for the sex chromosomes. At late anaphase or telophase, X₁ and X₂ join end-to-end but form spindle fiber connections to only one of the poles of the metaphase II spindle, leading to one daughter cell without X chromosomes and one with both X₁ and X₂. An attempt is made to explain sex chromosome pairing and orientation on the basis of a telocentric organization of meiotic chromosomes. The apparent differences in the kinetic organization of mitotic and meiotic chromosomes in Heteroptera are discussed.     

The founder effect theory: quantitative variation and mdg-1 mobile element polymorphism in experimental populations of Drosophila melanogaster

The chromosomes of 14 specimens of the genus Reithrodon from three different localities of Argentina and two localities of Uruguay were studied using G-and C-banding techniques. Specimens of Uruguay showed a karyotype of 2n=28 chromosomes having a large metacentric X, and a telocentric Y chromosome. This karyotype is very similar to that recently described in a sample from southern Brazil, differing only in the nature of the Y chromosome, which is metacentric in the Brazilian form. All specimens from Argentina showed a 2n=34 karyotype, differing from the Brazilian karyotype by two centric fusions, an acquisition of chromosome material, and at least one pericentric inversion, and by the telocentric nature of both the X and the Y chromosomes. G-and C-banding suggest that the metacentric gonosomes in the Brazilian form resulted from a double autosomal-X-Y Robertsonian translocation. The Uruguayan cytotype is interpreted as derived from a hypothetical neo-X/Y₁Y₂ ancestral form by the secondary loss of the Y₁ chromosome. The karyotypic differences between the Brazilian-Uruguayan and the Argentinian forms afford evidence of species differentiation. It is proposed to assign the former to Reithrodon typicus, and the later to R. auritus.