Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Using the biomass-ratio and idiosyncratic hypotheses to predict mixed-species litter decomposition

Background and Aims

Microsporogenesis leading to monosulcate pollen grains has already been described for a wide range of monocot species. However, a detailed study of additional callose deposition after the completion of the cleavage walls has been neglected so far. The study of additional callose deposition in monosulcate pollen grain has gained importance since a correlation between additional callose deposition and aperture location has recently been revealed.

Methods

Microsporogenesis is described for 30 species belonging to eight families of the monocots: Acoraceae, Amaryllidaceae, Alstroemeriaceae, Asparagaceae, Butomaceae, Commelinaceae, Liliaceae and Xanthorrhoeaceae.

Key Results

Five different microsporogenesis pathways are associated with monosulcate pollen grain. They differ in the type of cytokinesis, tetrad shape, and the presence and shape of additional callose deposition. Four of them present additional callose deposition.

Conclusions

In all these different microsporogenesis pathways, aperture location seems to be linked to the last point of callose deposition.     

Two New Species of Tylenchidae,Basiroides nortoni n. sp. and Tylenchus hageneri n. sp. (Nematoda: Tylenchida)

Basiroides nortoni n. sp. and Tylenchus hageneri n. sp. from soil around roots of corn from Ollie, Iowa, are described and illustrated. B. nortoni n. sp. has a posterior vulva, a long posterior uterine branch and an arcuate tail with pointed terminus. T. hageneri n. sp. has four lines in the lateral field, coarse transverse cuticular striae along the whole body, and a filiform tail slightly shorter than vulva-anus distance.     

Selenoproteins in Archaea and Gram-positive bacteria

Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis.     

Entomopathogenic Nematodes for Control of Insect Pests Above and Below Ground with Comments on Commercial Production

Entomopathogenic nematodes (EPNs) have been utilized in classical, conservation, and augmentative biological control programs. The vast majority of applied research has focused on their potential as inundatively applied augmentative biological control agents. Extensive research over the past three decades has demonstrated both their successes and failures for control of insect pests of crops, ornamental plants, trees and lawn and turf. In this paper we present highlights of their development for control of insect pests above and below ground. The target insects include those from foliar, soil surface, cryptic and subterranean habitats. Advances in mass-production and formulation technology of EPNs, the discovery of numerous efficacious isolates/strains, and the desirability of reducing pesticide usage have resulted in a surge of commercial use and development of EPNs. Commercially produced EPNs are currently in use for control of scarab larvae in lawns and turf, fungus gnats in mushroom production, invasive mole crickets in lawn and turf, black vine weevil in nursery plants, and Diaprepes root weevil in citrus in addition to other pest insects. However, demonstrated successful control of several other insects, often has not lead to capture of a significant share of the pesticide market for these pests.     

Homo floresiensis: a cladistic analysis

The announcement of a new species, Homo floresiensis, a primitive hominin that survived until relatively recent times is an enormous challenge to paradigms of human evolution. Until this announcement, the dominant paradigm stipulated that: 1) only more derived hominins had emerged from Africa, and 2) H. sapiens was the only hominin since the demise of Homo erectus and Homo neanderthalensis. Resistance to H. floresiensis has been intense, and debate centers on two sets of competing hypotheses: 1) that it is a primitive hominin, and 2) that it is a modern human, either a pygmoid form or a pathological individual. Despite a range of analytical techniques having been applied to the question, no resolution has been reached. Here, we use cladistic analysis, a tool that has not, until now, been applied to the problem, to establish the phylogenetic position of the species. Our results produce two equally parsimonious phylogenetic trees. The first suggests that H. floresiensis is an early hominin that emerged after Homo rudolfensis (1.86 Ma) but before H. habilis (1.66 Ma, or after 1.9 Ma if the earlier chronology for H. habilis is retained). The second tree indicates H. floresiensis branched after Homo habilis.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

Meloidogyne polycephannulata n. sp. (Nematoda: Meloidogynidae), a root-knot nematode parasitizing carrot in Brazil

Meloidogyne polycephannulata n. sp. is described from specimens collected from an area cultivated with carrot cv. Brasilia, in the city of Rio Paranaíba, in the region of Alto Paranaíba, Minas Gerais State, Brazil. The perineal pattern of the female is circular to ovoid with a high dorsal arch that has widely spaced, coarse annulations. The lateral field may have a deep furrow separating the dorsal and ventral arches. The medial lips are short and wide, whereas the lateral lips are large and triangular. The female stylet is 15-16 μm long with wide knobs, distinctly divided by an indentation in the center. Its tip is slightly curved dorsally. The excretory pore opens 34-65 μm from the anterior end. Females retain eggs and second-stage juveniles in their body cavity, similar to that of the cyst-forming nematodes. Males are 1.3-1.7 mm long and have a high head cap that is rounded and slopes posteriorly. The labial disc is fused to the medial lips. The head region has several irregular annulations that are similar in appearance to the first or second body annules that are likewise irregular, making the head region appear to be extremely large. The stylet of the male is 21-24 μm long; it is slender, and has small, rounded knobs, that are distinctly indented medially and appear heart-shaped. The shaft has several tiny projections throughout its length. Mean second-stage juvenile length is 411.7 μm. The juvenile head cap is elevated, the medial lips are small, and the lateral lips are elongate to triangular-shaped. The head region has several short, incomplete and irregular transverse annulations. The juvenile stylet is 14-23 μm long with small, rounded, and sloping knobs. The thin tail ends with a short hyaline portion that is variable in size (16-26 μm) and with a small, rounded tip. Isozyme profiles of esterases from Meloidogyne javanica show 3 strong bands (SB) at Rm 46, 59, and 66; profiles of M. polycephannulata n. sp. show a SB at Rm 47 and a weak band (WB) at Rm 52; M. petuniae has two SB at Rm 44 and 53; M. phaseoli has a SB at 53, 58, and 64 Rm; M. brasilensis has three SB at Rm 40, 58, and 66 and a WB at Rm 71; M. pisi has a SB at Rm 40, 60, and 64 and two WB at 46 and 50 Rm. Data from sequencing the 18S rDNA region of M. polycephannulata n. sp. confirms that it is different from M. arabicida, M. arenaria, M. ethiopica, M. incognita, M. javanica, M. paranaensis, and M. thailandica. Sequence identity among these eight species ranged between 85 to 93.4%. Meloidogyne polycephannulata n. sp. reproduces very well on carrot and tomato; poorly on pepper; and not at all on cotton, peanut, tobacco, watermelon, and sweet corn.     

A newly-identified lineage of Schistosoma

Because of their role in causing schistosomiasis, flukes of the genus Schistosoma are the best known of all digeneans. The genus has traditionally been divided into four familiar species groups. Here we report on three poorly known species of Schistosoma, one of which, Schistosoma hippopotami, is known from the hippopotamus, one of which is provisionally identified as Schistosoma edwardiense, another hippo parasite, and a third that has not previously been described. All were collected from freshwater snails obtained from Lake Edward, western Uganda, the type locality for both known hippo schistosomes. The three different kinds of schistosome cercariae differ from one another in size, and all are readily differentiated by their long tail stems from the cercariae of human-infecting species. Furthermore, each was recovered from a different genus of snail host, Biomphalaria sudanica, Bulinus truncatus or Ceratophallus natalensis. Molecular analysis, based on 8350 bases of combined nuclear and mitochondrial DNA, groups these three long tail-stem cercariae into a well supported clade that does not associate with any of the recognised species groups. The placement of this clade, basal to all African species plus several Asian species, suggests that there has been an ancient association between Schistosoma and hippos. This new African Schistosoma clade advocates the need for further modification of the traditional species group-based classification. Two of the four species groups are paraphyletic. It also suggests that Schistosoma has been remarkably plastic with respect to adapting to snail hosts-three distantly related genera of planorbid snails have been exploited by worms within a single clade. Finally, it adds a new layer of complexity to deciphering the origins of Schistosoma, often considered to be African but recently challenged as being Asian. In the late Cenozoic the distribution of hippo species straddled both Africa and Asia and they may have provided a means for the introduction of blood flukes to Africa.     

Acquisition and Distribution of Nematodes in Irrigation Waterways of the Columbia Basin in Eastern Washington

The primary source of plant parasitic nematodes in irrigation waterways in the Columbia Basin Project of eastern Washington is irrigation runoff returned into the irrigation system. This has contributed to the rapid spread of plant parasitic nematodes observed during eight years of study.     

Conserved regulation of the Caenorhabditis elegans labial/Hox1 gene ceh-13

Caenorhabditis elegans contains a set of six cluster-type homeobox (Hox) genes that are required during larval development. Some of them, but unlike in flies not all of them, are also required during embryogenesis. It has been suggested that the control of the embryonic expression of the worm Hox genes might differ from that of other species by being regulated in a lineal rather than a regional mode. Here, we present a trans-species analysis of the cis-regulatory region of ceh-13, the worm ortholog of the Drosophila labial and the vertebrate Hox1 genes, and find that the molecular mechanisms that regulate its expression may be similar to what has been found in species that follow a regulative, non-cell-autonomous mode of development. We have identified two enhancer fragments that are involved in different aspects of the embryonic ceh-13 expression pattern. We show that important features of comma-stage expression depend on an autoregulatory input that requires ceh-13 and ceh-20 functions. Our data show that the molecular nature of Hox1 class gene autoregulation has been conserved between worms, flies, and vertebrates. The second regulatory sequence is sufficient to drive correct early embryonic expression of ceh-13. Interestingly, this enhancer fragment acts as a response element of the Wnt/WG signaling pathway in Drosophila embryos.     

gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila

glial cells missing (gcm) is the primary regulator of glial cell fate in Drosophila. In addition, gcm has a role in the differentiation of the plasmatocyte/macrophage lineage of hemocytes. Since mutation of gcm causes only a decrease in plasmatocyte numbers without changing their ability to convert into macrophages, gcm cannot be the sole determinant of plasmatocyte/macrophage differentiation. We have characterized a gcm homolog, gcm2. gcm2 is expressed at low levels in glial cells and hemocyte precursors. We show that gcm2 has redundant functions with gcm and has a minor role promoting glial cell differentiation. More significant, like gcm, mutation of gcm2 leads to reduced plasmatocyte numbers. A deletion removing both genes has allowed us to clarify the role of these redundant genes in plasmatocyte development. Animals deficient for both gcm and gcm2 fail to express the macrophage receptor Croquemort. Plasmatocytes are reduced in number, but still express the early marker Peroxidasin. These Peroxidasin-expressing hemocytes fail to migrate to their normal locations and do not complete their conversion into macrophages. Our results suggest that both gcm and gcm2 are required together for the proliferation of plasmatocyte precursors, the expression of Croquemort protein, and the ability of plasmatocytes to convert into macrophages.     

Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

Impacts of diclidophorid monogenean infections on fisheries in Japan

Many monogeneans are pathogenic to economically important fish in Japan. However no other monogenean is comparable with the diclidophorids, Heterobothrium okamotoi and Neoheterobothrium hirame, on the scale of impacts they inflict on Japanese fisheries. The shared importance of the two monogenean infections lies in their pathogenicity, fecundity and tolerance to chemical treatment. Heterobothrium okamotoi infects the gills and wall of the branchial cavity of the tiger puffer, Takifugu rubripes (Tetraodontidae), which is widely cultured in western Japan. The main presenting signs of infected fish are anaemia and extensive necrosis caused by adult worms. This monogenean deposits long strings of eggs, which reach lengths of almost 3m. Egg entanglement with the mesh of culture nets increases the chance of hatched larvae encountering susceptible fish. The oncomiracidium maintains infectivity for up to 4 days after hatching. Hydrogen peroxide is the only commercially available chemical able to control the infection, but can only kill immature worms on the gills. Neoheterobothrium hirame infects the gills and wall of the buccal cavity of the Japanese flounder, Paralichthys olivaceus (Paralichthyidae). Since the first known occurrence of this monogenean in 1993, the species has been recorded from almost all areas where the host is distributed. Neoheterobothrium hirame has the potential to produce 781 eggs per day at 20 degreeC. In the western Sea of Japan, wild young-of-the-year flounder became infected in early summer, followed by a sharp increase in prevalence in late summer. By late summer, juvenile flounder have nearly disappeared from the area, strongly suggesting that N. hirame is responsible for mortality of young fish. This is in good agreement with the recent decline in the local flounder population. Neoheterobothrium hirame has also been considered the causative agent of anaemia among wild Japanese flounder since the late 1990s.     

Xenopsylla spp. (Siphonaptera: Pulicidae) in murid rodents from the Canary Islands: An update

The geographical and host distributions of Xenopsylla fleas parasitizing murid rodents on the Canary Islands have been reported. Three Xenopsylla species, X. cheopis, X. brasiliensis and X. guancha, have been detected on two rodents species, Mus musculus and Rattus rattus. X. guancha has been the most prevalent species detected, specifically on M. musculus, the most abundant rodent, but it has been detected only on three eastern islands, where the species is endemic. X. cheopis has been shown to be the most widely distributed species throughout the archipelago and the species most frequently found on R. rattus. X. brasiliensis has been shown to be the least prevalent Xenopsylla species, with the lowest geographical distribution on the Canary Islands and focused only on R. rattus. The detection of both X. cheopis and X. brasiliensis on the island of Lanzarote, and of X. guancha on the island of Fuerteventura and the islet of La Graciosa represents the first report of these species on those particular Canary Islands.     

Multiple host transfers,but only one successful lineage in a continent-spanning emergent pathogen

Emergence of a new disease in a novel host is thought to be a rare outcome following frequent pathogen transfers between host species. However, few opportunities exist to examine whether disease emergence stems from a single successful pathogen transfer, and whether this successful lineage represents only one of several pathogen transfers between hosts. We examined the successful host transfer and subsequent evolution of the bacterial pathogen Mycoplasma gallisepticum, an emergent pathogen of house finches (Haemorhous (formerly Carpodacus) mexicanus). Our principal goals were to assess whether host transfer has been a repeated event between the original poultry hosts and house finches, whether only a single host transfer was ultimately responsible for the emergence of M. gallisepticum in these finches, and whether the spread of the pathogen from east to west across North America has resulted in spatial structuring in the pathogen. Using a phylogeny of M. gallisepticum based on 107 isolates from domestic poultry, house finches and other songbirds, we infer that the bacterium has repeatedly jumped between these two groups of hosts but with only a single lineage of M. gallisepticum persisting and evolving in house finches; bacterial evolution has produced monophyletic eastern and western North American subclades.     

achaete, but not scute, is dispensable for the peripheral nervous system of Drosophila

The achaete-scute complex of Drosophila has been the focus of extensive genetic and developmental analysis. Of the four genes at this locus, achaete and scute appear to act redundantly to specify the peripheral nervous system. They share cis-regulatory elements and are co-expressed at the same locations. A mutation removing scute activity has been previously described; it causes a loss of some sensory bristles. Thus, when Scute is absent, the activity of achaete allows formation of the remaining bristles. However, all existing achaete mutants are rearrangements affecting regulatory sequences common to both achaete and scute. To determine the level of redundancy between the two genes, we have used a P element approach to generate a null allele of achaete, which leaves scute and all cis-regulatory elements intact. We find that the peripheral nervous system of achaete null mutant larvae and imagos lacks any detectable phenotype. However, when the levels of Scute are limiting, then some sensory organs are missing in achaete mutant flies. achaete and scute are thought to have arisen from a duplication event about 100 Myr ago. The difference between achaete and scute null flies is surprising and raises the question of the retention of both genes during the course of evolution.     

Middle Pleistocene fossil Cercopithecidae from Asbole, Afar Region, Ethiopia

A sample of 117 fossil cercopithecids has been collected from the Middle Pleistocene site of Asbole, Afar Region, Ethiopia. A minimum of five species is present. There are two species of Cercopithecini, here recognized as cf. Chlorocebus aff. aethiops, and cf. Chlorocebus cf. patas. There are also two species of Papionini: Papio hamadryas ssp. indet. and Theropithecus oswaldi leakeyi. Finally, there is a single species of colobine present, Colobus sp. indet. The assemblage is chronologically constrained and is derived from sediments dated to approximately 600 ka. Within this sample Colobus sp. is by far the most common species present, outnumbering the other four species combined. The cercopithecid assemblage is most consistent with a woodland habitat, corroborating an earlier interpretation based on the non-primate fauna. Taxonomic, biogeographic, and evolutionary implications of the assemblage are also discussed.