Population analysis of white grubs (Coleoptera: Melolonthidae) throughout the Brazilian Pampa biome

The replacement of natural grassland by cultivated areas might favor the increase in abundance of some root-feeding species such as the white grubs, which may become a constraint for field crop production. This research aimed to assay the population density and geographical distribution of white grubs pest and other species in natural grassland and cultivated areas throughout the Brazilian Pampa biome. White grubs were sampled in 18 locations in both landscape use types and identified. Population density (number of larvae m^?2) was calculated for each recorded species and sorted within two groups (pest species and other species), compared between natural grasslands and cultivated areas, as well as among locations. A dendrogram to evaluate species similarity among locations was built based on combined data obtained from both landscape use types throughout the region. In total, 31 species were found in the Brazilian Pampa, and four of them are considered as crop pests: Diloboderus abderus (Sturm, 1826), Euetheola humilis (Burmeister, 1847), Lyogenys fusca (Blanchard, 1830), and Phyllophaga triticophaga Morón & Salvadori, 1998. The average population density of pest species in cultivated areas was less than five larvae m^?2, at most of locations. Some species had a wide geographical distribution (e.g. D. abderus and Cyclocephala modesta Burmeister), while other melolontids occurred at only one location. The knowledge of which white grub species are present in a field and its population densities assist farmers to take proper management decisions.     

Degree of plant root digestion by the larva of the beetle, Costelytra zealandica

The degree of plant root digestion by grass grubs (Costelytra zealandica) has been evaluated by scanning electron microscope examination of plant residues in faeces. Faecal pellets were examined from grubs fed on roots of pure plant species (perennial ryegrass, Lolium perenne, and white clover, Trifolium repens) under soil-free conditions, as well as from naturally occurring grubs on mixed pasture. There was considerable variation in the degree of digestion observed with different plant species. Clover root was more extensively digested with formation of partially digested xylem vessels, whereas ryegrass root appeared to undergo very little digestive breakdown. These results indicate that plant structural carbohydrates undergo limited digestion by the grass grub. Thus grass grubs may require a high turnover of dietary root material, which would explain the highly destructive effect of the larvae on pasture plants.     

White grubs,Holotrichia rustica and Holotrichia mucida (Coleoptera: Scarabaeidae) as pests of teak (Tectona grandis L. f.) seedlings

Abstract Field observations were carried out between 2002 and 2005 on bio‐ecology of white grubs infesting teak (Tectona grandis Linn. f.) seedlings at Ramdongari Forest Nursery, Forest Development Corporation of Maharashtra, Nagpur in central India. The study collected the data required for an integrated pest management strategy against the white grubs. The species damaging teak seedlings were Holotrichia rustica (Burmeister) and H. mucida Gyllenhal. The beetles (adults) of both these species began to emerge just after the pre‐monsoon showers and when the relative humidity had risen rapidly over several days, but before the first monsoon rains. While the adults fed and mated on naturally growing bushes of Ziziphus jujuba, Z. mauritiana, Z. xylopyra, Acacia leucophloea and A. catechu immediately after the emergence from the teak beds in the nursery, the immature stages (grubs) of both species fed on roots of the teak seedling, leading to plant death. This is the first report of (i) damage caused to teak seedlings by grubs of H. rustica and H. mucida, and (ii) of Z. jujuba and Z. mauritiana being hosts for the beetles of H. mucida and H. rustica. It is also the first detailed report on the field bio‐ecology of the H. rustica and H. mucida as major pests of teak. White grubs are among the toughest‐to‐manage pests of economic importance and information presented here on the pest incidence and damage of H. rustica and H. mucida is important to researchers and nursery field managers for the management of these pests on teak.     

Phylogeographic history of Patagonian lizards of the Liolaemus elongatus complex (Iguania: Liolaemini) based on mitochondrial and nuclear DNA sequences

In this study, we present a phylogeographic analysis of a group of lizards distributed in north‐western Patagonia, the Liolaemus elongatus complex. We sequenced 581 individuals for one mitochondrial gene (cytochrome‐b), and for a subset, we sequenced another mitochondrial gene (12S rRNA) and two nuclear genes: kinesin family member 24 (KIF24) and the anonymous nuclear locus LDAB1D. We estimated gene trees, mitochondrial and nuclear haploytpe networks, standard molecular diversity indices, genetic distances between lineages and Bayesian skyline plots. Our results provide evidence for recognition of seven species previously described within the L. elongatus complex: Liolaemus antumalguen, Liolaemus chillanensis, Liolaemus carlosgarini, Liolaemus burmeisteri, Liolaemus smaug, Liolaemus elongatus and Liolaemus crandalli, but we did not find sufficient evidence to support Liolaemus choique, Liolaemus shitan or Liolaemus sp. 6 as distinct species. We identified four candidate species (Liolaemus sp. 1, Liolaemus sp. 2, Liolaemus sp. 3 and Liolaemus sp. 7), and we discuss evolutionary processes that may have contributed to the origin of these lineages and their taxonomic and conservation implications.     

Synergism of imidacloprid and entomopathogenic nematodes against white grubs: the mechanism

Entomopathogenic nematodes and the chloronicotinyl insecticide, imidacloprid, interact synergistically on the mortality of third-instar white grubs (Coleoptera: Scarabaeidae). The degree of interaction, however, varies with nematode species, being synergistic for Steinernema glaseri (Steiner) and Heterorhabditis bacteriophora Poinar, but only additive for Steinernema kushidai Mamiya. The mechanism of the interaction between imidacloprid and these three entomopathogenic nematodes was studied in the laboratory. In vials with soil and grass, mortality, speed of kill, and nematode establishment were negatively affected by imidacloprid with S. kushidai but positively affected with S. glaseri and H. bacteriophora. In all other experiments, imidacloprid had a similar effect for all three nematode species on various factors important for the successful nematode infection in white grubs. Nematode attraction to grubs was not affected by imidacloprid treatment of the grubs. Establishment of intra-hemocoelically injected nematodes was always higher in imidacloprid-treated grubs but the differences were small and in most cases not significant. The major factor responsible for synergistic interactions between imidacloprid and entomopathogenic nematodes appears to be the general disruption of normal nerve function due to imidacloprid resulting in drastically reduced activity of the grubs. This sluggishness facilitates host attachment of infective juvenile nematodes. Grooming and evasive behavior in response to nematode attack was also reduced in imidacloprid-treated grubs. The degree to which different white grub species responded to entomopathogenic nematode attack varied considerably. Untreated Popillia japonica Newman (Coleoptera: Scarabaeidae) grubs were the most responsive to nematode attack among the species tested. Untreated Cyclocephala borealis Arrow (Coleoptera: Scarabaeidae) grubs showed a weaker grooming and no evasion response, and untreated C. hirta LeConte (Coleoptera: Scarabaeidae) grubs showed no significant response. Chewing/biting behavior was significantly increased in the presence of nematodes in untreated P. japonica and C. borealis but not in C. hirta and imidacloprid-treated P. japonica and C. borealis. Our observations, however, did not provide an explanation for the lack of synergism between imidacloprid and S. kushidai.     

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela

Key message

Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times.

Abstract

White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.     

Two new Pythium species from China based on the morphology and DNA sequence data

In an investigation of Pythium species in lawn grassland of south China, two new species, Pythium breve and P. baisense, were identified based on morphological characteristics and DNA sequence data. These two new species differ morphologically from the other Pythium species by the oogonia encompassed by many antheridia and with 1?C3 antheridia on the wavy and curved stalks. Furthermore, P. baisense with complexly lobed antheridial stalks differs from P. breve with antheridial stalks entwining the hyphae nearby the oogonia with several turns. Results of phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species based on the internal transcribed spacer region (ITS1, 5.8S, ITS2) and cytochrome c oxidase subunit I (COXI) gene sequences using maximum parsimony and Bayesian methods. These two new species are described and illustrated in detail.     

Serovar Diversity of Pathogenic Leptospira Circulating in the French West Indies

Background

Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.

Methods and Findings

Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.

Conclusions

The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.     

Developing a new molecular marker for aphid species identification: Evaluation of eleven candidate genes with species-level sampling

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized as a molecular marker for aphid species identification. However, this gene has sometimes resulted in misidentification because of low interspecific genetic divergences between some species pairs. In this study, to propose new molecular markers for the family Aphididae, we first screened 2289 sequences of 11 genes (COI, COII, CytB, ATP6, lrRNA, srRNA, ITS1, ITS2, EF1a, 18S, and 28S) collected from the GenBank. Among the 11 genes, ATP6 gene revealed the largest genetic divergence among congeneric species with the smallest divergence among conspecific individuals; in contrast, species pairs with low genetic divergences (< 1%) were not observed. Secondly, for statistically testing the usefulness of ATP6 gene in species identification, we analyzed genetic distances between all of the combinations of 32 individuals of 20 species for both COI and ATP6 genes. The ATP6 gene showed lower intraspecific (on average 0.08%) and higher interspecific (on average 8.28%) genetic distances than the COI gene (on average 0.19% and 6.24%, respectively) for the same pairs of individuals. This study corroborates the usefulness of the ATP6 gene as a new molecular marker that could improve the misidentification problems that are inherent with the COI gene.     

Analysis of Tiphia parasitoids preovipositional behaviors and of their scarab host defensive responses

Tiphia vernalis Rohwer and Tiphia popilliavora Rohwer were introduced as biocontrol agents against Japanese beetles (Popillia japonica Newman) and oriental beetles (Anomala orientalis Waterhouse). Studies have shown that under field conditions, T. vernalis parasitize Japanese beetles more often than oriental beetles. This study was done to understand how tiphiid wasps handle the two different host species and the influence of host defensive behaviors on the oviposition process of tiphiid wasps. The preovipositional behaviors performed by Tiphia wasps included: stinging, examining, moving soil, kneading, host-feeding, and host scraping. The frequency, sequence, and total time spent on each behavior before oviposition were scored and compared between two host species. The sequence and frequency of preovipositional behaviors performed by both Tiphia wasps did not show a difference between the two host species. However, female T. vernalis spent significantly longer time trying to sting oriental beetles than Japanese beetles in order to paralyze them. The time T. popilliavora spent on prestinging behaviors did not show a difference between Japanese and oriental beetles. The defensive behaviors performed by Japanese and oriental beetle grubs included: vigorous movements, rubbing their abdomen or head against the wasp’s abdomen, and biting at the attacking wasp. The frequency and total time spent on each defensive behavior was scored and compared between two host species. Overwintered, third instar oriental beetle grubs spent significantly longer time on defensive behaviors when they were attacked by T. vernalis which likely cause wasps to spend longer time trying to sting oriental beetle grubs. The active host resistance gained through behavioral defenses could make oriental beetle grubs less susceptible to T. vernalis attack than Japanese beetle grubs, especially under field conditions. Younger grubs attacked by T. popilliavora did not exhibit these differences.     

Species limits,phylogeographic and hybridization patterns in Neotropical leaf frogs (Phyllomedusinae)

The taxonomy of many species is still based solely on phenotypic traits, which is often a pitfall for the understanding of evolutionary processes and historical biogeographic patterns, especially between closely related species due to either phenotypic conservatism or plasticity. Two widely distributed Neotropical leaf frogs from the Phyllomedusa burmeisteri species group (P. burmeisteri and Phyllomedusa bahiana) constitute a paramount example of closely related species with relatively unstable taxonomic history due to a large phenotypic variation. Herein, we analysed ~260 individuals from 57 localities distributed across the range of the two species to contrast individual phenotypic with an integrative phylogenetic and phylogeographic multilocus approach. We aim to clarify species limits, investigate potential undocumented diversity and examine to what extent taxonomic uncertainties could lead to misleading hypotheses on phylogeographic and interspecific hybridization patterns. Our molecular analysis supports the recognition of the two currently defined species, providing evidences for one novel and highly divergent evolutionary unit within the range of P. burmeisteri, which encompasses its type locality (Rio de Janeiro city). Spatial patterns of genetic and the colour of the hidden areas of the thigh was not congruent, varying considerably both within and between populations of both species. Genetic data showed signs of admixture between both species but do not corroborate the previously inferred wide area of introgression based on the distribution of the intermediate phenotype. Our results suggest that phenotypic variation can result from local adaptations, geographic isolation and/or evolutionary processes and, thus, cannot be used to reliably diagnose P. burmeisteri and P. bahiana. Globally, this study underscores the need of a geographical broad sampling of widespread species and the combination of molecular and phenotypic data to delineate species limits and phylogeographic patterns in species with complex taxonomy.     

Ecological Characterization of White Grubs (Coleoptera: Melolonthidae) Community in Cultivated and Noncultivated Fields

Comparative studies on the density and diversity of white grubs community (Coleoptera: Melolonthidae) occurring in cultivated and noncultivated fields of the Planalto region of the state of Rio Grande do Sul, Brazil, are presented. Sampling was carried out in 23 municipalities during the 2009 and 2010 winter seasons. Cultivated and noncultivated fields were chosen in each locality. Melolontid larvae were collected for identification and counted to determine the population density. A mean of 12.9 larvae m^?2 were collected in cultivated areas against 10.5 larvae m^?2 in noncultivated areas. The latter were more diverse (H′?=?2.52) than cultivated areas (H′?=?2.26). Despite the high evenness index (J?=?0.75 noncultivated and J?=?0.74 cultivated), faunistic parameters indicated Cyclocephala flavipennis Arrow and Diloboderus abderus Sturm as an extremely dominant species in cultivated areas. These results showed that the population density of white grubs increases, and their community composition is affected in cultivated areas.     

Transferability of molecular markers from major legumes to Lathyrus spp. for their application in mapping and diversity studies

Lathyrus cicera L. (chickling pea) and L. sativus L. (grass pea) have great potential among grain legumes due to their adaptability to inauspicious environments, high protein content and resistance to serious diseases. Nevertheless, due to its past underused, further activities are required to exploit this potential and to capitalise on the advances in molecular biology that enable improved Lathyrus spp. breeding programmes. In this study we evaluated the transferability of molecular markers developed for closely related legume species to Lathyrus spp. (Medicago truncatula, pea, lentil, faba bean and lupin) and tested the application of those new molecular tools on Lathyrus mapping and diversity studies. Genomic and expressed sequence tag microsatellite, intron-targeted amplified polymorphic, resistance gene analogue and defence-related gene markers were tested. In total 128 (27.7 %) and 132 (28.6 %) molecular markers were successfully cross-amplified, respectively in L. cicera and L. sativus. In total, the efficiency of transferability from genomic microsatellites was 5 %, and from gene-based markers, 55 %. For L. cicera, three cleaved amplified polymorphic sequence markers and one derived cleaved amplified polymorphic sequence marker based on the cross-amplified markers were also developed. Nine of those molecular markers were suitable for mapping in a L. cicera recombinant inbred line population. From the 17 molecular markers tested for diversity analysis, six (35 %) in L. cicera and seven (41 %) in L. sativus were polymorphic and discriminate well all the L. sativus accessions. Additionally, L. cicera accessions were clearly distinguished from L. sativus accessions. This work revealed a high number of transferable molecular markers to be used in current genomic studies in Lathyrus spp. Although their usefulness was higher on diversity studies, they represent the first steps for future comparative mapping involving these species.     

落叶松种间及无性系间ISSR鉴别技术的研究

采用ISSR分子标记技术对兴安、长白和日本落叶松种间以及不同无性系进行了鉴别。从49条引物中筛选出13条ISSR引物可以对落叶松种间和无性系间进行鉴别,特异条带个体的百分率为100%,该项技术为落叶松新品种以及良种的准确鉴别提供了新的途径和手段：其中5条引物在日本落叶松、兴安落叶松和长白落叶松不同位置扩增出特异谱带,作为种的鉴定的标准,有9条引物可以对落叶松种内不同无性系分别扩增出特异片段,进行无性系的鉴别。     

Diversity and Genetic Population Structure of Fungal Pathogens Infecting White Grub Larvae in Agricultural Soils

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.     

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Background

The number of species within the Malagasy genus Lepilemur and their phylogenetic relationships is disputed and controversial. In order to establish their evolutionary relationships, a comparative cytogenetic and molecular study was performed. We sequenced the complete mitochondrial cytochrome b gene (1140 bp) from 68 individuals representing all eight sportive lemur species and most major populations, and compared the results with those obtained from cytogenetic studies derived from 99 specimens.

Results

Interspecific genetic variation, diagnostic characters and significantly supported phylogenetic relationships were obtained from the mitochondrial sequence data and are in agreement with cytogenetic information. The results confirm the distinctiveness of Lepilemur ankaranensis, L. dorsalis, L. edwardsi, L. leucopus, L. microdon, L. mustelinus, L. ruficaudatus and L. septentrionalis on species level. Additionally, within L. ruficaudatus large genetic differences were observed among different geographic populations. L. dorsalis from Sahamalaza Peninsula and from the Ambanja/Nosy Be region are paraphyletic, with the latter forming a sister group to L. ankaranensis.

Conclusion

Our results support the classification of the eight major sportive lemur taxa as independent species. Moreover, our data indicate further cryptic speciation events within L. ruficaudatus and L. dorsalis. Based on molecular data we propose to recognize the sportive lemur populations from north of the Tsiribihina River, south of the Betsiboka River, and from the Sahamalaza Peninsula, as distinct species.     

Phylogenetic Relationships and Genetic Variation in Longidorus and Xiphinema Species (Nematoda: Longidoridae) Using ITS1 Sequences of Nuclear Ribosomal DNA

Genetic analyses using DNA sequences of nuclear ribosomal DNA ITS1 were conducted to determine the extent of genetic variation within and among Longidorus and Xiphinema species. DNA sequences were obtained from samples collected from Arkansas, California and Australia as well as 4 Xiphinema DNA sequences from GenBank. The sequences of the ITS1 region including the 3'' end of the 18S rDNA gene and the 5'' end of the 5.8S rDNA gene ranged from 1020 bp to 1244 bp for the 9 Longidorus species, and from 870 bp to 1354 bp for the 7 Xiphinema species. Nucleotide frequencies were: A = 25.5%, C = 21.0%, G = 26.4%, and T = 27.1%. Genetic variation between the two genera had a maximum divergence of 38.6% between X. chambersi and L. crassus. Genetic variation among Xiphinema species ranged from 3.8% between X. diversicaudatum and X. bakeri to 29.9% between X. chambersi and X. italiae. Within Longidorus, genetic variation ranged from 8.9% between L. crassus and L. grandis to 32.4% between L. fragilis and L. diadecturus. Intraspecific genetic variation in X. americanum sensu lato ranged from 0.3% to 1.9%, while genetic variation in L. diadecturus had 0.8% and L. biformis ranged from 0.6% to 10.9%. Identical sequences were obtained between the two populations of L. grandis, and between the two populations of X. bakeri. Phylogenetic analyses based on the ITS1 DNA sequence data were conducted on each genus separately using both maximum parsimony and maximum likelihood analysis. Among the Longidorus taxa, 4 subgroups are supported: L. grandis, L. crassus, and L. elongatus are in one cluster; L. biformis and L. paralongicaudatus are in a second cluster; L. fragilis and L. breviannulatus are in a third cluster; and L. diadecturus is in a fourth cluster. Among the Xiphinema taxa, 3 subgroups are supported: X. americanum with X. chambersi, X. bakeri with X. diversicaudatum, and X. italiae and X. vuittenezi forming a sister group with X. index. The relationships observed in this study correspond to previous genera and species defined by morphology.