Small frogs get their worms first: the role of nonodonate arthropods in the recruitment of Haematoloechus coloradensis and Haematoloechus complexus in newly metamorphosed northern leopard frogs, Rana pipiens, and woodhouse's toads, Bufo woodhousii

Studies on the life cycles and epizootiology of North American frog lung flukes indicate that most species utilize odonates as second intermediate hosts; adult frogs become infected by ingesting odonate intermediate hosts. Newly metamorphosed frogs are rarely infected with these parasites, predominantly because they are gape-limited predators that cannot feed on large intermediate hosts such as dragonflies. We examined the role of the frog diet and potential intermediate hosts in the recruitment of the frog lung fluke, Haematoloechus coloradensis, to metamorphosed northern leopard frogs (Rana pipiens), Woodhouse's toads (Bufo woodhousii), and bullfrogs (Rana catesbeiana) from western Nebraska. Because of the uncertain validity of H. coloradensis as a distinct species from Haematoloechus complexus, morphological characters of both species were reevaluated and the life cycles of both species were completed in the laboratory. The morphological data on H. coloradensis and H. coimplexus indicate that they differ in their oral sucker to pharynx ratio, uterine loop distribution, and placement of vitelline follicles. However, in terms of their life cycles, both species are quite similar in their use of physid snails as first intermediate hosts, a wide range of nonodonate and odonate arthropods as second intermediate hosts, and leopard frogs and toads as definitive hosts. These results indicate that H. coloradensis and H. complexus are generalists at the second intermediate host level and might be able to infect newly metamorphosed leopard frogs and toads by using small nonodonate arthropods more commonly than other frog lung fluke species. Comparisons of population structure of adult flukes in newly metamorphosed leopard frogs indicate that the generalist nature of H. coloradensis metacercariae enables it to colonize young of the year leopard frogs more commonly than other Haematoloechus spp. that only use odonates as second intermediate hosts. In this respect, the generalist nature of H. coloradensis and H. complexus at the second intermediate host level is an avenue for the colonization of young of year frogs.     

Evolutionary avenues for, and constraints on, the transmission of frog lung flukes (Haematoloechus spp.) in dragonfly second intermediate hosts

Metacercariae survival patterns and their distribution in second intermediate odonate hosts were examined for 4 species of frog lung flukes. Surveys of aquatic larvae and recently emerged teneral dragonflies and damselflies indicated that prevalence and mean abundance of Haematoloechus spp. metacercariae were significantly lower in teneral dragonflies than larval dragonflies, while there was no significant difference in prevalence or mean abundance of Haematoloechus spp. metacercariae among larval and teneral damselflies. Experimental infections of dragonflies indicated that metacercariae of Haematoloechus coloradensis and Haematoloechus complexus were located in the head, thorax, and branchial basket of dragonflies, whereas metacercariae of Haematoloechus longiplexus and Haematoloechus parviplexus were restricted to the branchial basket of these hosts. Metacercariae of H. coloradensis, H. complexus, and H. longiplexus infected the head, thorax, and abdomen of damselflies, but these insects were resistant to infection with H. parviplexus. Subsequent metamorphosis experiments on experimentally infected dragonflies indicated that most metacercariae of H. longiplexus were lost from the branchial basket during metamorphosis, but most metacercariae of H. coloradensis, H. complexus, and H. parviplexus survived dragonfly metamorphosis. These observations suggest that the observed ecological host specificity of H. longiplexus in semiterrestrial leopard frogs may be due to few metacercariae of H. longiplexus reaching these frogs in a terrestrial environment. Because of the uncertain validity of Haematoloechus varioplexus as a distinct species from its synonym H. parviplexus, their morphological characters were reevaluated. The morphological data on H. varioplexus and H. parviplexus indicate that they differ in their acetabulum length and width, ovary shape, testes length, and egg length and width. Experimental infections of plains leopard frogs, northern leopard frogs, and bullfrogs with worms from bullfrogs indicate that the synonymy of H. parviplexus with H. varioplexus is not warranted, and that these flukes are distinct species, i.e., H. parviplexus in bullfrogs and H. varioplexus in plains leopard frogs and northern leopard frogs.     

Morphological and molecular description of Haematoloechus meridionalis n. sp. (Digenea: Plagiorchioidea: Haematoloechidae) from Rana vaillanti brocchi of Guanacaste, Costa Rica.

An undescribed species of Haematoloechus inhabits the lungs of Rana vaillanti in northwestern Costa Rica. The new species is most similar morphologically to H. medioplexus, having a very small, but well-developed, ventral sucker and lacking extracecal uterine loops, and apparently was mis-identified previously as H. medioplexus in Rana palmipes from Colombia. It differs from H. medioplexus, notably by (1) the shape of the oral sucker, which is elliptical in H. meridionalis and spherical in H. medioplexus; (2) the posterior extent of the vitelline fields, which terminate at the same level in H. medioplexus, but always at different levels in H. meridionalis; (3) the position of the testes, which are immediately posterior to the seminal receptacle and are close together in the new species and far from the seminal receptacle and separated from each other in H. medioplexus; and (4) the location of the genital pore, which is ventral to the cecal bifurcation in the new species and ventral to the pharynx in H. medioplexus. In addition, the new species is unique among members of Haematoloechus by having flaps of tissue on the interior margins of the ventral sucker. The new species shows almost 5% sequence divergence from H. medioplexus in the variable D1 region of the 28s rDNA, complementing the morphological differences.     

Haematoloechus danbrooksi n. sp. (Digenea: Plagiorchioidea) from Rana vaillanti from Los Tuxtlas,Veracruz, Mexico

Haematoloechus danbrooksi n. sp. from the lungs of Rana vaillanti in Veracruz state, Mexico, was found. The new species is most similar morphologically to H. medioplexus, H. parviplexus, and H. meridionalis in having a ventral sucker less than half the diameter of the oral sucker. It differs from these species by the extension of the vitellaria, which are shorter in the new species, and in the shape of the tegumental spines, which are blunt in the new species. It differs from all other known species of Haematoloechus in the distribution of the uterine loops that are arranged diagonally and that present several short, extracecal. longitudinal loops in the postacetabular region. The new species shows 1.2% sequence divergence in partial 28S sequence with respect to H. medioplexus, 1.1% with H. parviplexus. and 2.5% with H. meridionalis, sequence divergences complementing the morphological differences.     

Phylogenetic and biogeographical relationships among some holarctic frog lung flukes (Digenea: Haematoloechidae).

A phylogenetic study of 8 North American and European species of frog lung flukes belonging to Haematoloechus was conducted using approximately 850 to 1,000 bases of the intemal transcribed spacer region (ITS 1 + 5.8S + ITS 2) and 1,250 bases of the large subunit (LSU) of the nuclear ribosomal DNA. Adequate phylogenetic resolution could not be obtained from 5.8S or ITS 2 data. Analysis of ITS 1 data produced 2 equally parsimonious trees that differed only in the position of Haematoloechus breviplexus relative to H. medioplexus and H. varioplexus. Single, identical trees were produced by analysis of both LSU sequence data and a data set comprised of all ITS and LSU data. All trees demonstrated 3 distinct evolutionary lineages within the Holarctic Haematoloechus examined. The results confirmed the taxonomic validity of H. abbreviatus and demonstrated that the presence or absence of extracecal uterine loops is not a character meaningful to the recognition of evolutionary lineages or differentiation of genera. Examination of ITS sequence data revealed almost no intraspecific variation within 5 species of Haematoloechus and demonstrated an approximately 150-base indel common to the North American H. longiplexus and the European H. asper. Two of 3 clades revealed by the phylogenetic analyses are comprised of both European and North American species, indicating that lineages of Haematoloechus arose before the breakup of Laurasia and radiated after Eurasia and North America split. Within each of 3 evolutionary lineages, members share similar patterns of arthropod host specificity distinct from patterns found in the other lineages. This suggests that second intermediate host specificity may be a trait that has been conserved through evolutionary time.     

Digeneans (Plathelminthes: Trematoda) of seven sympatric species of anurans from Los Tuxtlas,Veracruz, Mexico

Digeneans of sympatric Bufo marinus, B. valliceps, Hyla picta, H. ebraccata, Smilisca baudini, Rana berlandieri, and R. vaillanti from Los Tuxtlas, Veracruz, Mexico, are reported. A total of 7 adult trematode species and 3 unidentified metacercariae was found in the different host species examined: Gorgoderina attenuata, G. parvicava, Cephalogonimus americanus, Haematoloechus medioplexus, Glypthelmins californiensis, Mesocoelium monas and Langeronia macrocirrus. Prevalence, mean intensity and abundance of the trematodes in each host species are given.     

Haematoloechus medioplexus: light and electron microscope localization of dipeptidyl aminopeptidases I and II in the gastrodermis

The exopeptidases, dipeptidyl aminopeptidases I and II (EC 3.4.14.1 and EC 3.4.14.2, respectively) were demonstrated cytochemically at light and electron microscope levels in the gastrodermis of adult Haematoloechus medioplexus recovered from the lungs of naturally infected Rana pipiens. Lys-Ala-4-methoxy-beta-naphthylamide and Pro-Arg-4-methoxy-beta-naphthylamide were used as specific substrates. Reaction products from both enzymes were observed in the gastrodermis in what are believed to be vesicles analogous to secondary lysosomes. The localization of the enzymes in these vesicles coincides with previous reports of the distribution of acid phosphatase and esterase activities. The hydrolases are believed to function both intracellularly by autophagy and extracellularly by digestion of host globin in the cecal lumen. Cathepsin B activity was not observed following the cytochemical protocol used in this investigation.     

Molecular phylogeny of Haematoloechus Looss, 1899 (Digenea: Plagiorchiidae), with emphasis on North American species

Phylogenetic hypothesis of 23 populations corresponding to 18 species of the digenean Haematoloechus from America, Europe, and Africa, based on ribosomal DNA 28S partial sequences (approximately 890 bp), is presented. Genetic divergence between the in-group and the out-groups ranged from 9.7 to 14.5% and within the in-group, from 0.9 to 12.2%. Eight most parsimonious trees 569 steps long were obtained, with a consistency index of 72%. Groups in the tree are not congruent with those in previous classification schemes of species in the genus, based on a small number of morphological characters. For this subset of Haematoloechus species, plesiomorphic hosts are species of Rana, with 2 colonizations to other amphibian groups. African species appear to have diverged after the separation of Gondwana and Laurasia. Therefore, South American species should appear as the closest relatives of African species when included in the analysis. The evidence presented suggests an ancestral wide distribution of North American representatives of the group, followed by successive contraction, amplification, and fragmentation of ranges and speciation events as a result of the intense volcanic activity in the central part of Mexico since the late Tertiary, the drying climate of western and central United States and northwestern Mexico from the early Eocene to the Pleistocene, and the glaciation during the Pleistocene.     

Trematodes of the genus Haematoloechus (Digenea: Plagiorchiidae) from frogs in Korea.

Total 242 Rana nigromaculata and 9 R. catesbeiana were collected from the various localities in Korea from February 1989 to July 1991, and their lungs were examined. Five species in genus Haematoloechus, i.e., H. sibiricus japonicus (Yamaguti, 1936), H. nanchangensis Hsiung, 1934, H. variegatus (Rudolphi, 1819), H. lobatus (Seno, 1907), H. lobatus koreanus, were identified in this study. Among them, H. lobatus koreanus was a new subspecies and H. variegatus and H. lobatus were newly recorded from Korea.     

DNA probes for distinguishing Psychodopygus wellcomei from Psychodopygus complexus (Diptera:Psychodidae).

Genomic DNA fragments from males of Psychodopygus wellcomei were isolated and shown to be useful as sensitive diagnostic probes for positively separating individuals of this species from those of Ps. complexus. These two members of the Ps. squamiventris series are found sympatrically in foci of cutaneous leishmaniasis in the hill forests of southern Pará State. Of the two species, only Ps. wellcomei is thought to be an important vector of Leishmania braziliensis sensu stricto, but this is based on circumstantial evidence because of the difficulties of identifying female sandflies within the series. The diagnostic probes were isolated from a library of Ps. wellcomei built by ligating short fragments of Sau 3A-restricted, genomic DNA into the plasmid vector pUC 18. Differential screening of 1316.library clones with total genomic DNA of Ps. wellcomei and Ps. complexus identified 5 recombinants, with cross-hybridizing inserts of repetitive DNA, that showed strong specificity for Ps. wellcomei. As little as 0.4% of the DNA extracted from an individual sandfly (= ca. 0.5 nanograms) was specifically detected. The diagnostic probes were used to identify as Ps. wellcomei a wild-caught female sandfly found infected with L. braziliensis s.s., providing only the second positive association between these two species.     

Maternally derived yolk testosterone enhances the development of the hatching muscle in the red-winged blackbird Agelaius phoeniceus

Hatching asynchrony in avian species often leads to the formation of a size hierarchy that places last-hatched nestlings at a significant disadvantage. The hatching muscle (musculus complexus) is responsible for breaking the shell during hatching and for dorsal flexion of the neck during begging. An increase in its strength in last-hatched nestlings could mitigate the effects of hatching asynchrony by reducing the time required for hatching or enhancing the effectiveness of begging for parentally delivered food or both. We have previously found that yolk testosterone concentration increases with laying order in the red-winged blackbird Agelaius phoeniceus. In this study, we investigated the hypothesis that yolk testosterone has anabolic effects on the development of the complexus, thereby influencing competition among asynchronously hatched nestlings. We found that both yolk testosterone concentration and relative complexus mass (complexus mass/nestling body mass) increased with laying order and that these two variables were positively correlated in both newly hatched nestlings and in two-day-old broods. Moreover, direct injections of testosterone into egg yolks resulted in an increase in relative complexus mass, while injections of flutamide, a testosterone antagonist, resulted in a decrease in relative complexus mass. Neither yolk testosterone concentration nor relative complexus mass differed between male and female nestlings.     

Fine structure of the retinal pigment epithelium of the mallard duck (Anas platyrhynchos)

As part of a comparative morphological study, the fine structure of the retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in the mallard (Anas platyrhynchos). In this species the RPE consists of a single layer of cuboidal cells which display numerous very deep basal (scleral) infoldings and extensive apical (vitreal) processes which enclose photoreceptor outer segments. The RPE cells are joined laterally by prominent basally-located tight junctions. Internally smooth endoplasmic reticulum is the most abundant cell organelle with only small amounts of rough endoplasmic reticulum present. Polysomes are abundant as are basally-located mitochondria which often displayed a ring-shaped profile. The cell nucleus is large and vesicular. Melanosomes are plentiful only within the apical processes of the RPE cells in the light-adapted state. Myeloid bodies are large and numerous and very often have ribosomes on their outer surface. Bruch's membrane (complexus basalis) shows a pentalaminate structure but with only a poorly represented central elastic lamina. Profiles of the choriocapillaris are relatively small and the endothelium of these capillaries while extremely thin facing the retinal epithelium is but minimally fenestrated.     

Intraspecific variation of Haematoloechus floedae Harwood, 1932 (Digenea: Plagiorchiidae), from Rana spp. in North and Central America

Haematoloechus floedae was originally described from the lungs of Rana catesbeiana in Texas, and later reported in Florida, in the United States. It was proposed to be synonymous with H. breviplexus, H. parviplexus, and H. varioplexus. We obtained specimens of H. floedae from Rana brownorum and R. vaillanti in Yucatán, Mexico; R. cf. forreri and R. taylori in Guanacaste, Costa Rica; and R. catesbeiana in Georgia and California. Some specimens were processed for morphological study; sequences of the 28S of the rDNA and the mitochondrial COI were obtained from several specimens of each population. Phylogenetic analysis of molecular data indicates studied populations constitute a single taxon, different from H. varioplexus, H. breviplexus, and H. parviplexus. Some morphological characters remain constant among populations of H. floedae, and are useful for differentiating this species. These include the oral sucker-pharynx ratio, oral sucker-ventral sucker ratio, ovary and testes shape, extension of uterine longitudinal loops, and extension of vitelline follicles (in fully developed worms). Sequence homogeneity among populations of H. floedae suggests a recent spread, perhaps due to the introduction of R. catesbeiana for culture to the western United States, southeast Mexico, and Costa Rica.     

Functional morphology of female goblin spider genitalia (Arachnida: Araneae: Oonopidae) with notes on fertilization in spiders

The genital structures of most spiders are poorly investigated in respect of their functional morphology because the traditional taxonomic practice is to inspect slide-mounted genitalia only. The present study describes the female genitalia of three members belonging to the megadiverse haplogyne spider family Oonopidae by means of histological serial sections, scanning electron microscopy, and X-ray ultramicroscopy. The female genitalia of Neoxyphinus ogloblini, Dysderina sp., and Heteroonops spinimanus are complex and might have evolved under sexual selection by cryptic female choice. However, there is no direct evidence for cryptic female choice in these species based on the results of the present study. In N. ogloblini and Dysderina sp., spermatozoa and secretion are stored in a large receptaculum. Highly elongated gland cells filled with secretory vesicles extend over the receptaculum of N. ogloblini. In addition, sperm are present in the uterus internus of female N. ogloblini and Dysderina sp. The location of fertilization is still unknown for most spiders. One female of Dysderina sp. had sperm in the uterus and ovary strongly suggesting that fertilization in this species takes place in the ovary. An anterior sclerite with attached muscles should serve females to lock the uterus externus during copulation as suggested for other oonopids. The male palp of N. ogloblini shows a simple embolus whereas the embolus of Dysderina sp. is more complicated and accompanied by a cork-screw-shaped conductor. Females of H. spinimanus have an anterior sclerite in which thread-like gland ducts lead. The chitinized posterior diverticulum shows peculiar papillae in its anterior wall. The exact location of sperm storage in H. spinimanus remains unknown since spermatozoa were not present in the anterior sclerite and the posterior diverticulum. The anterior sclerite might be used to lock the uterus externus similar to N. ogloblini and Dysderina sp. H. spinimanus was previously suggested to be parthenogenetic and a male has only been recently associated with this species. The male was not investigated for this study.     

Patterns of infection by lungworms, Rhabdias ranae and Haematoloechus spp., in northern leopard frogs: a relationship between sex and parasitism

We examined a population of northern leopard frogs to determine whether sex biases in investment in immunity, previously reported for this host species under controlled exposures to lung nematodes, is predictive of patterns of parasitism in nature. We examined Rhabdias ranae and Haematoloechus spp. infections in 74 breeding adult, 28 non-breeding adult, and 53 juvenile frogs. Contrary to our predictions, R. ranae prevalence and mean abundance were higher in breeding female frogs (prevalence: 39.4%, abundance: 3.05 +/- 0.85) than on breeding males (prevalence: 26.0%, abundance: 1.17 +/- 0.52), although no sex bias was observed among non-breeding adults or juvenile frogs. Female frogs also carried larger R. ranae worms, on average, than did males (females: 6407.38 microm +/- 153.80; males: 5198 microm +/- 131.09), regardless of age or breeding condition. We observed no sex-linked patterns of parasitism by Haematoloechus spp. worms in either adult or juvenile frogs. Alternative hypotheses, such as differences among sexes in the selection of thermal clines for hibernation, may explain the observed female bias in parasitism by nematode lungworms in nature and, thus, need to be considered.     

6种蝙蝠精子储存现象的组织学观察

精子储存是蝙蝠的生殖对策之一,有些种类的蝙蝠能将成熟精子在交配前(雄性)或交配后(雌性)储存在附睾或者输卵管及子宫内数月。采用冰冻切片和苏木精-伊红(H.E)染色法,观察了6种蝙蝠的睾丸、附睾、卵巢、输卵管和子宫,发现5种蝙蝠有精子储存现象。另外,本文还讨论了不同生殖对策蝙蝠的地理分布。     

New species of Oswaldocruzia (Nematoda: Molineoidae), new species of Rhabdias (Nematoda: Rhabdiasidae), and other helminths in Rana cf. forreri (Anura: Ranidae) from Costa Rica

Oswaldocruzia costaricensis n. sp. (Strongylida: Molineidae) from the intestines and Rhabdias savagei n. sp. (Rhabditida: Rhabdiasidae) from the lungs of Rana cf. forreri (Anura: Ranidae) are described and illustrated. Oswaldocruzia costaricensis represents the 77th species assigned to the genus and differs from the other Neotropical species in the genus by possessing a Type II bursa and long cervical alae. Rhabdias savagei represents the 47th species assigned to the genus and differs from other Neotropical species in the genus by possession of 4 lips and a postequatorial vulva. Rana cf. forreri was also found to harbor the trematodes, Haematoloechus parcivitellarius and Megalodiscus temperatus, the nematodes, Aplectana incerta, Aplectana itzocanensis, Cosmocerca podicipinus, Foleyellides striatus, Subulascaris falcaustriformis, and a larva of the nematode Brevimulticaecum sp. Cosmocerca panamaensis is considered to be a synonym of Cosmocerca podicipinus.     

Isoforms of myosin and actin in human, monkey and rat myometrium. Comparison of pregnant and non-pregnant uterus proteins

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.