Sibling Species Status Research Articles

Malaria vector sibling species distribution in different endemic districts of Punjab, India.

Malaria transmission in Punjab, India is mainly seasonal with variation in its endemicity that may be due to varying vector behaviour in different areas of the state, primarily attributed to the existence of sibling species complexes among the vector species. So far there is no report regarding the existence of malaria vectors sibling species in the state of Punjab, therefore, the present study was planned to investigate the status of sibling species of two main vectors of malaria viz. Anopheles culcifacies and Anopheles fluviatilis in different districts of Punjab. Mosquito collections were made through hand catch in the morning hours. Malaria vector species An. culicifacies and An. fluviatilis were morphologically identified and man hour density was calculated. Both the vector species were subjected to molecular assays for sibling species identification through amplification of D3 domain of 28S ribosomal DNA by allele-specific PCR. Four sibling species of An. culicifacies, were identified viz. A, B, C and E. Species A was identified from Bhatinda district, species B, C and E from. S.A.S. Nagar and species C from Hoshiarpur. Two sibling species S and T of An. fluviatilis were identified from districts S.A.S. Nagar and Rupnagar. Presence of four sibling species of An. culicifacies and two sibling species of An. fluviatilis in Punjab necessitates planning of longitudinal studies to ascertain their role in disease transmission so that appropriate interventions may be applied to achieve malaria elimination.

Cytotypes of the Simulium arcticum Species Complex in the American Pacific Northwest

The larval polytene chromosomes of 17 of the 22 known cytotypes of the Simulium arcticum Malloch complex were analyzed and compared at 59 collection sites in the Pacific Northwest of the United States. Cytotypes range from those that were broadly distributed, such as IIL-9, which was found at 25 different sites, to those that were found at only one or two sites, such as IIL-73·74. Cytotypes are “clustered” in a geographic sense, suggesting that once a new cytotype is described, if it is found again, it will be found nearby. The cytotype S. arcticum IIL-9 was found at 11 of the 25 sites where S. arcticum IIL-19 exists. Finally, based on its sibling species status and widespread distribution throughout the Pacific Northwest, Simulium brevicercum may be an early ancestor of the complex given that it occurs at 44 of the 59 sites sampled. The observations and conclusions of this research are scientifically important because previous research suggests that reproductive isolation may have begun among these cytotypes at these sites, and unless their locations are reported, they cannot be studied further. Future work should incorporate a multiomic approach to study the putative initial divergences within this complex.

Alterations and Interchange of Morphometric Characters in Different Life Cycle Stages with Reference to Genomic Variations of Anopheles subpictus (Diptera; Culicidae) Sibling Species Complex in Sri Lanka.

The species complex of the mosquito Anopheles subpictus is designated by the sibling species A–D, depending on morphological characters of life cycle stages and variations in polytene chromosomes. However, morphological aberrations in the life cycle stages make the identification of sibling species uncertain and imprecise. The objective of the present study is to determine the suitability of morphological variations of sibling species and their genomic variations to identify the sibling species status of an An. subpictus population in Sri Lanka. Life cycle stages of larvae, pupal exuviae, and adults were examined for previously reported distinctive morphological features. Five nuclear and mitochondrial genome regions, including the Internal transcribed spacer 2 (ITS2) region, D3 region, white gene, cytochrome c oxidase I (COI), and Cytochrome b (Cyt-b), were sequenced and analyzed for variations. The eggs changed their distinct sibling morphological characters during metamorphosis (89.33%). The larvae, pupal exuviae, and adult stages showed deviation from their sibling characters by 26.10%, 19.71%, and 15.87%, respectively. However, all the species from the analysis shared two distinct sequence types for all regions, regardless of the morphological variations. In conclusion, the An. subpictus sibling species complex in Sri Lanka is not identifiable using morphological characters due to variations, and the genomic variations are independent from the morphological variations.

Differentiating sibling species of Zeugodacus caudatus (Insecta: Tephritidae) by complete mitochondrial genome.

Zeugodacus caudatus is a pest of pumpkin flowers. It has a Palearctic and Oriental distribution. We report here the complete mitochondrial genome of the Malaysian and Indonesian samples of Z. caudatus determined by next-generation sequencing of genomic DNA and determine their taxonomic status as sibling species and phylogeny with other taxa of the genus Zeugodacus. The whole mitogenome of both samples possessed 37 genes (13 protein-coding genes-PCGs, 2 rRNA and 22 tRNA genes) and a control region. The mitogenome of the Indonesian sample (15,885bp) was longer than that of the Malaysian sample (15,866bp). In both samples, TΨC-loop was absent in trnF and DHU-loop was absent in trnS1. Molecular phylogeny based on 13 PCGs was concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with the two samples of Z. caudatus forming a sister group and the genus Zeugodacus was monophyletic. The Malaysian and Indonesian samples of Z. caudatus have a genetic distance of p=7.8% based on 13 PCGs and p=7.0% based on 15 mitochondrial genes, indicating status of sibling species. They are proposed to be accorded specific status as members of a species complex.

Mitochondrial Genome Supports Sibling Species of Angiostrongylus costaricensis (Nematoda: Angiostrongylidae).

Angiostrongylus costaricensis is a zoonotic parasitic nematode that causes abdominal or intestinal angiostrongyliasis in humans. It is endemic to the Americas. Although the mitochondrial genome of the Brazil taxon has been published, there is no available mitochondrial genome data on the Costa Rica taxon. We report here the complete mitochondrial genome of the Costa Rica taxon and its genetic differentiation from the Brazil taxon. The whole mitochondrial genome was obtained from next-generation sequencing of genomic DNA. It had a total length of 13,652 bp, comprising 36 genes (12 protein-coding genes—PCGs, 2 rRNA and 22 tRNA genes) and a control region (A + T rich non-coding region). It is longer than that of the Brazil taxon (13,585 bp). The larger mitogenome size of the Costa Rica taxon is due to the size of the control region as the Brazil taxon has a shorter length (265 bp) than the Costa Rica taxon (318 bp). The size of 6 PCGs and the start codon for ATP6, CYTB and NAD5 genes are different between the Costa Rica and Brazil taxa. Additionally, the two taxa differ in the stop codon of 6 PCGs. Molecular phylogeny based on 12 PCGs was concordant with two rRNA, 22 tRNA and 36 mitochondrial genes. The two taxa have a genetic distance of p = 16.2% based on 12 PCGs, p = 15.3% based on 36 mitochondrial genes, p = 13.1% based on 2 rRNA genes and p = 10.7% based on 22 tRNA genes, indicating status of sibling species. The Costa Rica and Brazil taxa of A. costaricensis are proposed to be accorded specific status as members of a species complex.

Identification of sibling species status of Anopheles culicifacies breeding in polluted water bodies in Trincomalee district of Sri Lanka

BackgroundAnopheles culicifacies s.l., the major vector of malaria in Sri Lanka, is known to breed in clean and clear water. However, recent findings have confirmed breeding from waste water bodies in urban and semi-urban areas. No study has been conducted to identify whether it is vector or non-vector siblings. The objective of the study was to identify the sibling species status of An. culicifacies s.l.MethodsAnopheles culicifacies s.l. adult samples (reared from larvae) were obtained from the Padavisiripura Entomological team attached to Tropical and Environmental Diseases and Health Associates (TEDHA) Malaria Elimination Programme in Trincomalee District. The collected mosquito specimens were processed for the extraction of genomic DNA individually. The PCR amplifications were carried out using different primer combinations for differentiating species A from D, species B from C, species B from E, and species B, C, and E from each other. The results obtained from the gel electrophoresis were compared with the marker and band sizes of 359 bp, 248 bp, 95 + 248 bp, 166 + 359 bp and 178 + 248 bp were used to compare the sibling species A, B, C, D and E, respectively.ResultsThe molecular biological identification of the field caught An. culicifacies s.l. samples observed that only 13.34 % (4/30) was represented sibling species B. About 86.66 % (26/30) of the samples were An. culicifacies sibling species E. This study also provided evidence that An. culicifacies E was able to breed in a wide range of breeding habitats. This is the first time that An. culicifacies E breeding in waste water was confirmed by a molecular method. Malaria control programmes in most parts of the country focus on rural communities as a result of bio-ecology of mosquitoes. Therefore, unusual breeding habitats, such as waste water collections, may mislead the current vector controlling programmes.ConclusionsThese results reconfirm that An. culicifacies s.l. has adapted to breed in a wide range of water bodies, including waste water collections. Since the majority of mosquitoes sampled belong to sibling species E, this may adversely affect the current malaria elimination programme and new strategies should be adopted to control malaria vectors breeding in these unusual breeding habitats in Sri Lanka.

Spodoptera frugiperda (Lepidoptera: Noctuidae) host-plant variants: two host strains or two distinct species?

The moth Spodoptera frugiperda is a well-known pest of crops throughout the Americas, which consists of two strains adapted to different host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. Though morphologically indistinguishable, they exhibit differences in their mating behavior, pheromone compositions, and show development variability according to the host-plant. Though the latter suggest that both strains are different species, this issue is still highly controversial because hybrids naturally occur in the wild, not to mention the discrepancies among published results concerning mating success between the two strains. In order to clarify the status of the two host-plant strains of S. frugiperda, we analyze features that possibly reflect the level of post-zygotic isolation: (1) first generation (F1) hybrid lethality and sterility; (2) patterns of meiotic segregation of hybrids in reciprocal second generation (F2), as compared to the meiosis of the two parental strains. We found a significant reduction of mating success in F1 in one direction of the cross and a high level of microsatellite markers showing transmission ratio distortion in the F2 progeny. Our results support the existence of post-zygotic reproductive isolation between the two laboratory strains and are in accordance with the marked level of genetic differentiation that was recovered between individuals of the two strains collected from the field. Altogether these results provide additional evidence in favor of a sibling species status for the two strains.Electronic supplementary materialThe online version of this article (doi:10.1007/s10709-015-9829-2) contains supplementary material, which is available to authorized users.

Phylogenetic Analysis of Anopheles (Cellia) subpictus Grassi Using rDNA-ITS2 Sequence

Anopheles subpictus Grassi is one of the most abundant malaria vector mosquitos in Indian subcontinent especially in post monsoon months. This taxon has been speculated to be composed of four sibling species recognised using morphological and cytogenetic parameters provisionally named A, B, C and D. One of the sibling species ‘B’ has well documented status as a vector of malaria parasite in the Oriental region. Molecular phylogenetic analysis of this mosquito was done to discern sibling/cryptic species using internal transcribed spacer 2 region of the nuclear ribosomal DNA (rDNA-ITS2). An. culicifacies sibling species A, a member of Myzomia series, was used as an out-group to root the trees. In the present analysis rDNA-ITS2 region was PCR amplified, sequenced and the sequences obtained were then subjected to phylogenetic analysis with the sequences already present in the sequence database GenBank. Multiple sequence alignment was performed using ClustalX and further manually annotated in MEGA 4. Initial analysis suggested extreme 3′ end sequence divergence within different populations. Phylogenetic analysis of this spacer was done using maximum parsimony, maximum likelihood, and distance matrix—neighbour joining methods performed with bootstrapped dataset that were executed in DNAPARS, DNAML, DNADIST and neighbour programs in Phylip software package respectively. Phylogenetic trees produced were similar in topology but differed in bootstrap support. Results support the division of this taxon into at least two major clades differing in their sequence composition and product size that are recommended to be renamed as An. subpictus inland form and An. subpictus coastal form. Differences include insertions/deletions, transitions and transversions; especially one large 3′ end deletion event. The sibling species status of An. subpictus has been analysed critically.

Variations in susceptibility to common insecticides and resistance mechanisms among morphologically identified sibling species of the malaria vector Anopheles subpictus in Sri Lanka.

BackgroundAnopheles subpictus s.l., an important malaria vector in Sri Lanka, is a complex of four morphologically identified sibling species A-D. Species A-D reportedly differ in bio-ecological traits that are important for vector control. We investigated possible variations that had not been reported previously, in the susceptibility to common insecticides and resistance mechanisms among the An. subpictus sibling species.MethodsAdult An. subpictus were collected from localities in four administrative districts in the dry zone of Sri Lanka. Single female isoprogeny lines were established and sibling species status determined according to reported egg morphology. World Health Organization's standard protocols were used for insecticide bioassays and biochemical assays to determine insecticide susceptibility and resistance mechanisms. Susceptibility of mosquitoes was tested against DDT (5%), malathion (4%), deltamethrin (0.05%) and λ-cyhalothrin (0.05%). Biochemical basis for resistance was determined through assaying for esterase, glutathione-S-transferase and monooxygenase activities and the insensitivity of acetycholinesterase (AChE) to propoxur inhibition.ResultsAll sibling species were highly resistant to DDT. However there were significant differences among the sibling species in their susceptibility to the other tested insecticides. Few species A could be collected for testing, and where testing was possible, species A tended to behave more similarly to species C and D than to B. Species B was more susceptible to all the tested insecticides than the other sibling species. This difference may be attributed to the predominance of species B in coastal areas where selection pressure due to indoor residual spraying of insecticides (IRS) was lower. However there were significant differences between the more inland species C and D mainly towards pyrethroids. Higher GST activities in species C and D might have contributed to their greater DDT resistance than species B. Malathion resistance in both species C and D may be caused by elevated GST activity and an altered insensitive target site in AChE. In addition, a carboxylesterase based malathion resistance mechanisms was also detected in species C and D. Elevated esterase levels in species C and D might have contributed to the low levels of pyrethroid resistance. However an absence of elevated activity of monooxygenases in species B, C and D indicates that monooxygenases are unlikely to be the cause of this partial resistance to pyrethroids.ConclusionsThe differences in insecticide susceptibility and insecticide resistance mechanism shown by An. subpictus sibling species are important considerations for developing the malaria control and eradication program in Sri Lanka. Similar studies on species complexes of other anopheline vectors of malaria are necessary for effective malaria control worldwide. The differential susceptibility findings are also consistent with most, if not all, morphologically identified An. subpictus species B in Sri Lanka belonging to the An. sundaicus complex. There is a need therefore to develop molecular techniques that can be used to differentiate morphologically similar anopheline species in field conditions for more effective vector control.

Molecular evidence for the presence of malaria vector species a of the Anopheles annularis complex in Sri Lanka

BackgroundAnopheles annularis s.l. is a wide spread malaria vector in South and Southeast Asia, including Sri Lanka. The taxon An. annularis is a complex of two sibling species viz. A and B, that are differentiated by chromosome banding patterns and ribosomal gene sequences in India. Only species A is reported to be a malaria vector in India while the occurrence of sibling species in Sri Lanka has not been documented previously.FindingsAnopheline larvae were collected at a site in the Jaffna district, which lies within the dry zone of Sri Lanka, and reared in the laboratory. Emerged adults were identified using standard keys. DNA sequences of the D3 domain of 28S ribosomal DNA (rDNA) and the internal transcribed spacer-2 (ITS-2) of the morphologically identified An. annularis were determined. BLASTn searches against corresponding An. annularis sequences in GenBank and construction of phylogenetic trees from D3 and ITS-2 rDNA sequences showed that the Sri Lankan specimens, and An. annularis s.l. specimens from several Southeast Asian countries were closely related to species A of the Indian An. annularis complex.ConclusionsThe results show the presence of the malaria vector An. annularis species A in Sri Lanka and Southeast Asia. Because An. annularis vectors have been long associated with malaria transmission in irrigated agricultural areas in the Sri Lankan dry zone, continued monitoring of An. annularis populations, and their sibling species status, in these areas need to be integral to malaria control and eradication efforts in the island.

Genetic evidence for malaria vectors of the Anopheles sundaicus complex in Sri Lanka with morphological characteristics attributed to Anopheles subpictus species B

BackgroundAnopheles subpictus sensu lato, a widespread malaria vector in Asia, is reportedly composed of four sibling species A - D. Mosquitoes morphologically identified as belonging to the Subpictus complex were collected from different locations near the east coast of Sri Lanka, and specific ribosomal DNA sequences determined to validate their taxonomic status.MethodsAnopheles subpictus s.l. larvae and blood-fed adults were collected from different locations in the Eastern province and their sibling species status was determined based on published morphological characteristics. DNA sequences of the D3 domain of 28 S ribosomal DNA (rDNA) and the internal transcribed spacer -2 (ITS-2) of mosquitoes morphologically identified as An. subpictus sibling species A, B, C and D were determined.ResultsPhylogenetic analysis based on D3 domain of rDNA resulted in two clades: one clade with mosquitoes identified as An. subpictus species A, C, D and some mosquitoes identified as species B, and another clade with a majority of mosquitoes identified as species B with D3 sequences that were identical to Anopheles sundaicus cytotype D. Analysis of ITS-2 sequences confirmed a close relationship between a majority of mosquitoes identified as An. subpictus B with members of the An. sundaicus complex and others identified as An. subpictus B with An. subpictus s.l.ConclusionsThe study suggests that published morphological characteristics are not specific enough to identify some members of the Subpictus complex, particularly species B. The sequences of the ITS-2 and D3 domain of rDNA suggest that a majority that were identified morphologically as An. subpictus species B in the east coast of Sri Lanka, and some identified elsewhere in SE Asia as An. subpictus s.l., are in fact members of the Sundaicus complex based on genetic similarity to An. sundaicus s.l. In view of the well-known ability of An. sundaicus s.l. to breed in brackish and fresh water and its proven ability to transmit malaria in coastal areas of many Southeast Asian countries, the present findings have significant implications for malaria control in Sri Lanka and neighbouring countries.

Anopheles culicifacies breeding in brackish waters in Sri Lanka and implications for malaria control

BackgroundAnopheles culicifacies is the major vector of both falciparum and vivax malaria in Sri Lanka, while Anopheles subpictus and certain other species function as secondary vectors. In Sri Lanka, An. culicifacies is present as a species complex consisting of species B and E, while An. subpictus exists as a complex of species A-D. The freshwater breeding habit of An. culicifacies is well established. In order to further characterize the breeding sites of the major malaria vectors in Sri Lanka, a limited larval survey was carried out at a site in the Eastern province that was affected by the 2004 Asian tsunami.MethodsAnopheline larvae were collected fortnightly for six months from a brackish water body near Batticaloa town using dippers. Collected larvae were reared in the laboratory and the emerged adults were identified using standard keys. Sibling species status was established based on Y-chromosome morphology for An. culicifacies larvae and morphometric characteristics for An. subpictus larvae and adults. Salinity, dissolved oxygen and pH were determined at the larval collection site.ResultsDuring a six month study covering dry and wet seasons, a total of 935 anopheline larvae were collected from this site that had salinity levels up to 4 parts per thousand at different times. Among the emerged adult mosquitoes, 661 were identified as An. culicifacies s.l. and 58 as An. subpictus s.l. Metaphase karyotyping of male larvae showed the presence of species E of the Culicifacies complex, and adult morphometric analysis the presence of species B of the Subpictus complex. Both species were able to breed in water with salinity levels up to 4 ppt.ConclusionsThe study demonstrates the ability of An. culicifacies species E, the major vector of falciparum and vivax malaria in Sri Lanka, to oviposit and breed in brackish water. The sibling species B in the An. subpictus complex, a well-known salt water breeder and a secondary malaria vector in the country, was also detected at the same site. Since global warming and the rise in sea levels will further increase of inland brackish water bodies, the findings have significant implications for the control of malaria in Sri Lanka and elsewhere.

Identification of a large hybrid zone between sympatric sibling species of Triatoma dimidiata in the Yucatan peninsula, Mexico, and its epidemiological importance

Triatoma dimidiata is one of the major Chagas disease vectors, with an extensive diversity in its morphology, habitat, and level of domiciliation. Molecular studies based on the internal transcribed spacer 2 (ITS-2) have subdivided this species into four potential taxonomic groups. Using both ITS-2 and cytochrome B markers, we confirmed the sibling species status of ITS-2 Group 3 and detected an apparent sympatry of ITS-2 Groups 2 and 3 in the Yucatan peninsula, Mexico. Here we examine the geographic distribution of T. dimidiata ITS-2 genotypes in the region and compare their egg production and Trypanosoma cruzi infection rates, as indicators of biological differences between groups. PCR genotyping of large natural populations showed an extensive sympatry of Groups 2 and 3 in most of the peninsula, often within the same house. We also detected a large proportion of individuals displaying ITS-2 sequences from both Groups 2 and 3, suggesting hybridization. Analysis of ITS-2 genotype frequencies indicated a strong departure from Hardy–Weinberg equilibrium in female hybrids, but not in males, due to a large heterozygote deficit. These results suggest random mating between ITS-2 Groups 2 and 3 combined with reduced viability and/or survival in female hybrids. This and other factors may allow for the maintenance of distinct ITS-2 Groups 2 and 3 populations despite high hybrid frequencies. Importantly, T. cruzi infection was much higher in hybrids compared to ITS-2 Groups 2 and 3 individuals, but all three genotypes appeared to seasonally infest houses in a similar manner in the region. These findings warrant further studies on T. dimidiata taxonomy and its epidemiologic implications.

Species-Specificity of Chemical Signals: Silk Source Affects Discrimination in a Wolf Spider (Araneae: Lycosidae)

Female spiders deposit chemical cues that elicit male courtship behavior with silk. These cues are often assumed to be species-specific although male spiders may court in response to chemical cues of closely-related species. We used behavioral assays to test the extent of species discrimination of female chemical cues by male Schizocosa ocreata, a wolf spider (Lycosidae). Discrimination, expressed as relative courtship intensity of males, varied significantly with phylogenetic distance. Males did not discriminate between female cues of conspecifics and a sibling species, S. rovneri. Courtship response was intermediate for another species within the ocreata clade and not different from control for spiders outside the clade. These findings support the sibling species status of S. ocreata and S. rovneri, and also suggest the composition of female chemical signals is conserved across closely related wolf spider species.

Seasonal variation as a determinant of population structure in rotifers reproducing by cyclical parthenogenesis

Monogonont rotifers live in habitats that display extensively variation in both biotic and abiotic components. Much of this variation is seasonal and therefore predictable for a given pond or lake. In 1972, King proposed one physiological and two genetic models presenting alternative modes of adaptation to this temporal variation. Our purpose in the present paper is to review and evaluate how our knowledge of the seasonal structure of rotifer populations has changed in the past 25 years. Seasonal changes in clone frequencies have been reported from three studies of natural populations using electrophoretic analysis of isozymes. In one of these studies there was evidence for substantial temporal overlap of multilocus genotypes suggesting that these clones were broad-niched generalists. By contrast, both the genetic and ecological analyses in the other two studies support a non-overlap model in which clonal groups are composed of narrow-niched specialists that undergo seasonal succession. In both of these studies the clonal groups appear to have achieved the status of sibling species, a phenomenon that we conclude is probably common in monogonont rotifers. Strong competition promotes reproductive isolation between successive groups of seasonal specialists. The existence of this competition has been inferred from natural populations and demonstrated by studies in the laboratory. Also required, and also supported by field observations, is a temporal separation of periods of mictic (sexual) reproduction. A final requirement of the nonoverlap model is seasonal variation in the timing of resting egg hatching. That is, clones established from hatching of resting eggs must enter a physiologically appropriate habitat if they are to increase in number and achieve a competitive advantage. Unfortunately, we still have little information on this topic. Finally, we present the results of a study analyzing the effects of variation in the mictic ratio (i.e., the relative frequency of mictic females) on the adaptive structure of rotifer populations. Mixis may shift the balance between costs and benefits of specialization thereby producing seasonally specialized populations that overlap in space but not time. Life history patterns may therefore provide fundamental insights on the adaptation of rotifers to the extensive temporal variation in their environments.

The Simulium metallicum species complex (Diptera: Simuliidae) in Latin America: a cytological study

Eleven members of the Simulium metallicum complex from Central America and South America are described using the polytene chromosome banding pattern of S. metallicum A as standard. The members are B from Mexico and Guatemala, C from Colombia, D and E from Venezuela, F from Panama, G from Costa Rica, H from Mexico, Guatemala, and Panama, I from Mexico and Guatemala, and J and K from Panama. A cytophylogeny separates the members into three lineages. H, I, J, and K form one lineage, separated from the standard by eight rearrangement steps. D and E form a second lineage on the basis of six fixed inversions in common. Three floating inversions characterize the common progenitor of C and G, which together form a third lineage. B is independently derived from the standard and separated from it by seven rearrangement steps. F is very close to A. Six cytotypes (A, B, H, I, J, and K) appear cytologically to be reproductively isolated and are considered sibling species. One cytotype, E, is a sibling species by virtue of cytological criteria, and possible ecological and physiological factors. The remaining four members (C, D, F, and G) are cytotypes requiring additional evidence to confirm sibling species status. Sibling A is provisionally considered to represent S. metallicum s.s.; sibling H may be S. horacioi Okazawa &Onishi. The status of members within the complex as vectors of Onchocerca volvulus is discussed.

Genetic variation and divergence of the sibling pair of cyprinid fishes, Notropis cornutus and N. chrysocephalus

Horizontal starch gel electrophoresis was used to estimate the amount of genetic divergence between Notropis cornutus and N. chrysocephalus. Measures of genetic identity (1) and distance ( D) were 0.881 and 0.127 ± 0.055 (s.e.), respectively. These estimates correspond more closely to the sibling species status of these taxa than other previously reported estimates. Notropis cornutus was found to be significantly more variable than N. chrysocephalus electrophoretically and morphologically. Assuming the existence of an electrophoretic clock, the time of divergence was calculated to be roughly 1.9–2.5 million years. This estimate corresponds very closely to a previously hypothesized late Pliocene divergence.

Multiple function of pteridines in Drosophila I. Specific fluorescent patterns in three sibling species

Three sibling species of Drosophila, D. melanogaster, D. simulans and D. mauritiana, still without complete reproductive isolation, show specific fluorescent patterns. The difference is so striking that a tubular lamp of 365 nm U.V. light and a binocular are sufficient for routine observations. Besides those differences, it has been concluded from the comparison with totally different species like Drosophila busckii and D. littoralis that the basic pattern formations of the three species are broadly the same. Observations of behaviour under U.V. light suggest that the visual stimulation is integrated in communication in addition to olfactory, aural and tactile stimuli. Our preliminary results suggest that fluorescence is widely present in insects, perhaps even in arthropods in general. Such fluorescent patterns can perhaps shed a new light upon the status of sibling species. The results suggest that fluorescence is dependent on redox-sensitive pteridines.

Esterase variation at three loci in meat ants.

The meat ant (Iridomyrmex purpureus) occurs in a number of color forms, with uncertain taxonomic status. Gel electrophresis of meat ant extracts, followed by nonspecific esterase staining, reveals several zones of activity. Allelic variation at three loci is proposed to account for variation in some of these zones. Two of the loci (Es-1, Es-2) appear to have recessive null alleles, whose frequencies have been estimated by the method of maximum likelihood. Geographic variation in allele frequency is attributed to behavioral and geographic subdivision of the population. Apparent disturbances in segregation ratios and deviations from Hardy-Weinberg equilibrium can be accounted for if it is argued that some nests contain more than one queen. Differences in gene frequency between sympatric populations of the red and blue forms of I. purpureus are observed, confirming their reproductive is isolation and sibling species status.