Comparative analyses of effective population size within and among species: ranid frogs as a case study

It has recently become practicable to estimate the effective sizes (N(e) ) of multiple populations within species. Such efforts are valuable for estimating N(e) in evolutionary modeling and conservation planning. We used microsatellite loci to estimate N(e) of 90 populations of four ranid frog species (20-26 populations per species, mean n per population = 29). Our objectives were to determine typical values of N(e) for populations of each species, compare N(e) estimates among the species, and test for correlations between several geographic variables and N(e) within species. We used single-sample linkage disequilibrium (LD), approximate Bayesian computation (ABC), and sibship assignment (SA) methods to estimate contemporary N(e) for each population. Three of the species-Rana pretiosa, R. luteiventris, and R. cascadae- have consistently small effective population sizes (<50). N(e) in Lithobates pipiens spans a wider range, with some values in the hundreds or thousands. There is a strong east-to-west trend of decreasing N(e) in L. pipiens. The smaller effective sizes of western populations of this species may be related to habitat fragmentation and population bottlenecking.     

Phylogenetic analysis of Hepatozoon species (Apicomplexa: Adeleorina) infecting frogs of Nova Scotia, Canada, determined by ITS-1 sequences

Species of Hepatozoon are apicomplexan parasites infecting tetrapod vertebrates and hematophagous arthropods. Two species, Hepatozoon catesbianae and Hepatozoon clamatae, have been described inhabiting the erythrocytes of bullfrogs and green frogs. A number of characteristics typically used to distinguish between members of this genus are shared between these 2 species, prompting speculation as to whether or not these organisms are in fact distinct species. To test the species distinction, bullfrogs and green frogs were captured at various sites across Nova Scotia, blood samples were collected, and DNA was extracted from samples containing parasites. The internal transcribed spacer 1 (ITS-1) from geographically diverse samples of both species was amplified by PCR, sequenced, and analyzed. ITS-1 sequences from the 2 species revealed single-nucleotide polymorphisms at 6 sites. Phylogenetic analysis of these molecular data and cytopathological features place isolates of each species in separate monophyletic groups. Comparison of the ITS-1 sequences between isolates from Nova Scotia and Ontario revealed that ITS-1 sequences of H. catesbianae from a previous study were mischaracterized as being those of H. clamatae. Phylogenetic data based on molecular variation and cytopathological features from this study provide the strongest evidence to date supporting the distinction between these 2 species.     

The water frogs of Greece: Bioacoustic evidence for a new species

The structure of the mating call of lake frogs (referred to as R. ridibunda) from 16 populations in Greece was analyzed for local variation using multivariate statistics. The populations of Thrace and of the island of Samothraki form a group giving the same type of mating call, whereas the mating call of the other populations differs in the degree of temperature dependence of four parameters, and specifically in the number of pulses/pulse group and pulse groups/call. Discriminant functions distinguish even single call series with a probability of 97%, intermediate mating calls are absent, and there is a significant, but slight differentiation of external morphological characters. These results have strong taxonomic implications. We conclude that the lake frogs of Greece comprise two species. The mating call of the lake frogs from Thrace resembles in all parameters that of the Rana ridibunda in the terra typica restricta (Guryev, CIS). Accordingly, the lake frogs of eastern Greece belong to R. ridibunda. The mating call of these lake frogs consists of 20 pulses/pulse group and of 7 pulse groups/call on the average. Most of Greece is inhabited by the second taxon, Rana balcanica sp. n. Its mating call is characterized by 27 pulses/pulse group and 4 pulse groups/call on the average. The two species in Greece do not differ with respect to coloration and size, but several standardized indices vary significantly: body length/digitus primus length; body length/callus internus length; body length/snout-eye distance; body length/tympanum diameter; tibia length/callus internus length; maximal head width/snout-eye distance.     

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.     

Hepatozoon species of the timber rattlesnake in northern Florida: description of a new species, evidence of salivary gland oocysts, and a natural cross-familial transmission of an Hepatozoon species

Two species of Hepatozoon, i.e., H. sauritus and H. horridus n. sp., were present in 1 of 8 timber rattlesnakes, Crotalus horridus. The narrow gamonts of H. sauritus are 15.0-19.0 x 3.5-5.0 microm, with LW 58-86 microm2 and L/W 3.2-4.7, with a narrow, rounded anterior end. The spherical to slightly ovoid oocysts produce ovoid to elongate sporocysts, 21-43 x 12-24 microm, L/W 1.20-2.7, containing on average 22.1 (10-34) sporozoites. This is the first report of a natural cross-familial transfer of a Hepatozoon species. Gamonts of H. horridus n. sp. are 13.0-17.0 x 4.0-6.0 microm, with LW 63-102 microm2 and L/W 2.6-4.0, and have broadly rounded ends. The gamont cytoplasm is vacuolated. The spherical to ovoid oocysts form spherical to elongate sporocysts 14-45 x 11-25 microm, L/W 1.0-2.3, producing an average of 13.0 (8-21) sporozoites. The salivary gland in 1 of 5 mosquitoes dissected contained 1 mature oocyst.     

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

The dendritic cell receptor DC-SIGN discriminates among species and life cycle forms of Leishmania

Infection of dendritic cells by the human protozoal parasite Leishmania is part of its survival strategy. The dendritic cell receptors for Leishmania have not been established and might differ in their interactions among Leishmania species and infective stages. We present evidence that the surface C-type lectin DC-SIGN (CD 209) is a receptor for promastigote and amastigote infective stages from both visceral (Leishmania infantum) and New World cutaneous (Leishmania pifanoi) Leishmania species, but not for Leishmania major metacyclic promastigotes, an Old World species causing cutaneous leishmaniasis. Leishmania binding to DC-SIGN was found to be independent of lipophosphoglycan, the major glycoconjugate of the promastigote plasma membrane. Our findings emphasize the relevance of DC-SIGN in Leishmania-dendritic cell interactions, an essential link between innate and Leishmania-specific adaptive immune responses, and suggest that DC-SIGN might be a therapeutic target for both visceral and cutaneous leishmaniasis     

First evidence for photoperiodic regulation of the life cycle in a millipede species, Polydesmus angustus (Diplopoda: Polydesmidae)

The hypothesis that the periods of dormancy previously described in the millipede Polydesmus angustus may be photoperiodically induced diapauses was tested experimentally. In this species, biennial individuals exhibit two successive periods of dormancy: aestivation in the penultimate stadium (stadium VII) and reproductive dormancy in the adults, which emerge in autumn. It was first established that the reproductive dormancy is not a thermally controlled state of quiescence. When adults emerging in autumn were kept at 16 °C under natural photoperiod, their reproduction was delayed for several months in comparison with adults emerging in spring at similar temperatures. This indicates that the reproductive dormancy begins with a period of diapause. Further experiments provided evidence of a photoperiodic induction of the adult diapause. When millipedes were reared under short day length (L:D 12:12 h) throughout their development, they required more time to reproduce than millipedes reared under long day length (L:D 16:8 h) at the same temperatures. Photoperiod influenced reproduction in females, but no significant effects were detected in adult males. On the other hand, stadium VII was markedly longer at L:D 16:8 h than at L:D 12:12 h in both sexes, which strongly suggests that aestivation is also induced by photoperiod. However, the effects on the duration of stadium VII varied among individuals, some of which showed no response to long days. This study is the first to document photoperiodic regulation of the life cycle in the class Diplopoda, a trait common in other classes of terrestrial arthropods.     

Phylogenetic relationships among isolates of Cryptosporidium: evidence for several new species

Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.     

Cell cycle characteristics of crenarchaeota: unity among diversity

The hyperthermophilic archaea Acidianus hospitalis, Aeropyrum pernix, Pyrobaculum aerophilum, Pyrobaculum calidifontis, and Sulfolobus tokodaii representing three different orders in the phylum Crenarchaeota were analyzed by flow cytometry and combined phase-contrast and epifluorescence microscopy. The overall organization of the cell cycle was found to be similar in all species, with a short prereplicative period and a dominant postreplicative period that accounted for 64 to 77% of the generation time. Thus, in all Crenarchaeota analyzed to date, cell division and initiation of chromosome replication occur in close succession, and a long time interval separates termination of replication from cell division. In Pyrobaculum, chromosome segregation overlapped with or closely followed DNA replication, and further genome separation appeared to occur concomitant with cellular growth. Cell division in P. aerophilum took place without visible constriction.     

No evidence for melatonin-linked immunoenhancement over the annual cycle of an avian species

The winter immunoenhancement hypothesis associates long nights and increased exposure to melatonin with enhanced immune function in winter when resource availability is low and the chances of becoming ill are high. Thus, increased exposure to melatonin in the winter could be adaptive for species facing difficult winter conditions. This idea has found some support in studies of resident mammals. In birds, the link between day length and melatonin over the annual cycle is weaker, and contributions of melatonin to seasonal timing are unclear. Furthermore, many species, especially migrants, do not experience the most difficult conditions of their annual cycle in winter. In this study, we tested whether the winter immunoenhancement hypothesis holds in an avian species, the red knot Calidris canutus. We found that melatonin duration and amplitude varied significantly over the annual cycle with the highest values occurring in winter. However, peaks did not correspond to the winter solstice or with annual variation in immune function. Our findings do not support the winter immunoenhancement hypothesis in knots and question whether the idea that immune function should be bolstered in winter can be generalized to systems where winter is not the most difficult time of the year.     

Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.     

User needs analysis and development of priorities for life cycle impact assessment

The development of the LCIA programme of the UNEP/SETAC Life Cycle Initiative started with a global survey of LCA practitioners. There were 91 LCIA-specific responses from all global regions. Respondents gave an indication of how they use LCA with respect to both the stage of LCA that they base decisions on (LCI, LCIA or a combination of both) as well as the types of decisions which they support with LCA information. The issues requiring immediate attention within the UNEP SETAC Life Cycle Initiative identified from this User Needs analysis are the need for transparency in the methodology, for scientific confidence and for scientific co-operation as well as the development of a recommended set of factors and methodologies. Of interest is the fact that results from the different regions highlighted the need for different impact categories. Based on this information proposals were made for new impact categories to be included in LCA (and thus LCIA). The LCIA programme aims to enhance the availability of sound LCA data and methods and to deliver guidance on their use. More specifically, it aims to 1) make results and recommendations widely available for users through the creation of a worldwide accessible information system and 2) establish recommended characterisation factors and related methodologies for the different impact categories, possibly consisting of sets at both midpoint and damage level. The work of the LCIA programme of the UNEP/SETAC Life Cycle Initiative has been started within four task forces on 1) LCIA information system and framework, 2) natural resources and land use, 3) toxic impacts, and 4) transboundary impacts. All participants willing to contribute to these efforts are invited to contact the LCIA programme manager or to join the next LCIA workgroup meeting that will take place in at the world SETAC congress in Portland on Thursday 18 November 2004.     

Heterochronies in the cranial development of Asian tree frogs (Amphibia: Anura: Rhacophoridae) with different life histories

The development of bony skull was studied in four species of Asian tree frogs (Rhacophoridae) with different life histories: biphasic development with free larval stage and direct development. In biphasic rhacophorids the sequence of the appearance of cranial bones generally followed the generalized pattern of craniogenesis, which was described for most studied anurans. In contrast, direct-developing species displayed some heterochronies in the formation of skull bones, namely, the accelerated formation of the anlagen of jaw and suspensorium bones. The obtained results support that the embryonization in amphibians is regularly accompanied by a heterochronic repatterning of craniogenesis, rather similar in different phyletic groups.