Occurrence of Apristurus species in the Galicia Bank Seamount (NE Atlantic)

The aim of this study was to identify some of the Apristurus species by combining morphometric and genetic tools. Several specimens of the genus Apristurus were caught on the Galicia Bank Seamount (NE Atlantic), between 1460 and 1809 m depths, during a multidisciplinary survey carried out in 2011 within the framework of the INDEMARES Project. Morphometric and genetic analyses were conducted to aid the identification of the specimens collected. A total of 20 specimens were identified, of which 18 corresponded to Apristurus aphyodes (Nakaya and Stehmann, 1998 ), one to A. profundorum (Goode and Bean, 1896 ) and one to A. melanoasper Iglesias, Nakaya & Stehmann, 2004 . Genetic results based on the mtDNA COI sequences (682–690 bp fragment of the COI gene) support the identification of A. profundorum and A. melanoasper, with a bootstrap of 99 and 96%, respectively. The identification of A. aphyodes was also performed using a 499 bp fragment of the 16S mitochondrial gene. These are the first records of the Apristurus species from Galician waters, which extends their known area of distribution and provides more information on different biological and ecological aspects of this complex taxonomic group.     

Description of the larva of <Emphasis Type="Italic">Curteria</Emphasis> Southcott, 1961 (Acari,Parasitengona, Erythraeidae) with notes on biology and life cycle

The larvae of Curteria episcopalis (C.L. Koch, 1837) and Curteria southcotti Gabryś, 1992, are described based on specimens reared in the laboratory. Pedroerythraeus Haitlinger, 2004 is considered a junior synonym of Curteria. Data on habitat specificity and phenology of the species as well as on development of eggs into larvae are given.     

Simultaneous identification of Grapholita molesta Busck and Grapholita dimorpha Komai by PCR‐RFLP

Although several molecular diagnostic techniques are available for the identification of the apple‐feeding pests Grapholita molesta Busck and Grapholita dimorpha Komai, these pests are severely affecting apple orchards in Korea. These two pests may be misidentified or the available molecular diagnostic techniques may not facilitate the simultaneous identification of the morphological features of both species. In this study, we developed a multiplex assay for these two species using the polymerase chain reaction – restriction fragment length polymorphism (PCR‐RFLP) method. Sixty‐two specimens were collected from apples presumed infested with moth larvae and from pheromone traps from 2013 to 2014. Both species were identified morphologically, and a partial region of the cytochrome b gene was sequenced to design primers for PCR‐RFLP. Digestion profiles of G. molesta and G. dimorpha, using the Sau3A1 restriction enzyme, were characterized using three DNA fragments each for G. molesta (363 bp, 91 bp and 31 bp) and G. dimorpha (220 bp, 234 bp and 31 bp). The RFLP assay developed for both species in this study was more efficient and accurate than other currently used diagnostic assays and would be helpful to identify field‐collected specimens for pest control research.     

Association of insect life stages using DNA sequences: the larvae of Philodytes umbrinus (Motschulsky) (Coleoptera: Dytiscidae)

Abstract. Insect life stages are known imperfectly in many cases, and classifications are based often on only one or a few semaphoronts of a species. This is unfortunate as information in alternative life stages often is useful for scientific study. Although recent examples of DNA in taxonomy have emphasized the identification of indistinguishable species, such sequence data facilitate the association of life history stages and hold considerable promise in phylogenetic analysis, evolutionary studies, diagnostics, etc. These concepts are discussed here and an example is provided from diving beetles (Dytiscidae: Coleoptera). Three unknown larval specimens of an apparent species of Laccophilinae collected in Namibia were associated with the species Philodytes umbrinus (Motschulsky) using DNA sequence data. An 806-bp portion of the gene cytochrome oxidase I was sequenced from the unknown larvae. Several identified adult specimens of species of Laccophilinae from Namibia were also sequenced, including two P. umbrinus specimens and specimens from four Laccophilus Leach species. Additional species of Laccophilus from other areas of the world also were sequenced, as were specimens of Agabetes acuductus (Harris), Australphilus saltus Watts, Neptosternus boukali Hendrich & Balke and a species of Laccodytes Régimbart. Parsimony analysis resulted in two most parsimonious trees with the unknown larva unambiguously resolved in a group with both adult specimens of P. umbrinus (bootstrap value = 100%). The average pairwise p-distance between the unknown larva and adult P. umbrinus specimens averaged 0.09% (0–0.14%), compared with an average divergence between other conspecifics in the analysis of 0.24% (0–0.82%) and an overall average divergence between species of 13.49% (1.90–19.86%). Based on this, the unknown larvae were assigned to P. umbrinus. The larvae are diagnosed and described and their relationship with other Laccophilinae is discussed.     

Geometric morphometrics as a tool for identifying emperor fish (Lethrinidae) larvae and juveniles

The aim of this study was to evaluate the efficiency of geometric morphometrics for describing the body shape of fish larvae and juveniles, and identifying them to species, in comparison with traditional linear measurements. Species of emperor fishes (Perciformes: Lethrinidae, genus Lethrinus) were chosen as the model group, as the late larval and early juvenile stages in this genus are particularly difficult to identify. Forty‐five individuals of different species of Lethrinus were collected from the south‐western lagoon of New Caledonia between May 2005 and March 2006. The individuals were first identified to species by their partial cytochrome‐b gene sequence. They were then morphologically characterized using eight linear measurements and 23 landmarks recorded on digital photographs. Except for a small proportion of individuals, geometric morphometrics gave better results to distinguish the different species than linear measurements. A ‘leave one out’ approach confirmed the nearly total discrimination of recently settled Lethrinus genivittatus and Lethrinus nebulosus, whereas traditional identification keys failed to distinguish them. Therefore, geometric morphometrics is a promising tool for identifying fish larvae and juveniles to species. An effective approach would require building image databases of voucher specimens associated with their DNA barcodes. These images could be downloaded by the operator and processed with the specimens to be identified.     

Larvae of chigger mites Neotrombicula spp. (Acari: Trombiculidae) exhibited Borrelia but no Anaplasma infections: a field study including birds from the Czech Carpathians as hosts of chiggers

Chigger mites were collected from 1,080 wild birds of 37 species at Certak (Czech Republic), in the western Carpathian Mountains, from 29 July to 24 September 2005. The prevalence of infestation with chigger larvae was 7%. A total of 325 chigger specimens from 10 bird species was identified and three chigger species were found: Neotrombicula autumnalis, N. carpathica, and N. inopinata, the latter two species being reported on new hosts. Neotrombicula carpathica is reported in the Czech Republic for the first time. A total of 509 chigger larvae found on 79 host specimens were examined by polymerase chain reaction (PCR) for the presence of Borrelia burgdorferi s.l. DNA (fragments of the rrf (5S)—rrl (23S) intergenic spacer), and Anaplasma phagocytophilum DNA (epank1 gene). A fragment of specific Borrelia DNA was amplified through PCR in one sample, and the PCR product was further analyzed by reverse line blotting assay, whereby both genospecies of B. garinii and B. valaisiana were proved. This sample pooled five chigger larvae collected from one Sylvia atricapilla on 11 August 2005. No A. phagocytophilum DNA was amplified. We conclude that larvae of the genus Neotrombicula can be infected with Borrelia genospecies originated from their present or former hosts.     

The parasite fauna of <Emphasis Type="Italic">Arctogadus glacialis</Emphasis> (Peters) (Gadidae) from western and eastern Greenland

In all 155 specimens of the high Arctic codfish Arctogadus glacialis examined for metazoan parasites, 55 specimens were from southern and northern Baffin Bay, western Greenland, and 100 specimens from north-eastern Greenland and Scoresby Sound. A total of 20 parasite taxa were recorded. A new myxozoan Gadimyxa arctica was found in southern Baffin Bay and Scoresby Sound. The gadid myxozoan Zschokkella hildae, the digeneans Gonocerca phycidis and Lecithaster gibbosus, the gill copepod Haemobaphes cyclopterina and third-stage larvae of the nematodes Anisakis simplex and Hysterothylacium aduncum were found in Scoresby Sound only. The digenean Hemiurus levinseni and third-stage larvae of the nematode Contracaecum sp. were found at all four stations. The nematodes Ascarophis spp. were found at three stations. The parasite fauna of A. glacialis from Scoresby Sound was very similar to that of 50 specimens of the closely related Boreogadus saida from the same area.     

<Emphasis Type="Italic">Boreogadus saida</Emphasis> (Lepechin) (Gadidae): a review of its metazoan parasite fauna from Greenland,eastern Canada,Alaska and the Russian Arctic

The metazoan parasite fauna of 50 specimens of Boreogadus saida (Lepechin) (Gadidae) from eastern Greenland is very similar to those of previous studies of the parasite fauna of B. saida from Greenland, eastern Canada, Alaska and the Russian Arctic. The digeneans Hemiurus levinseni, Derogenes varicus and Lecithaster gibbosus and cestode larvae were found at most stations. Single specimens of the nematode larvae Anisakis simplex were found at four stations. A comparison of the distribution of the larvae of Contracaecum sp. and Hysterothylacium sp. is difficult due to a possible confusion of the two genera. Most of the metazoan parasites of B. saida are generalist species found in several fish families. Boreogadus saida acquires most of its endoparasites by eating pelagic crustaceans, mainly copepods and amphipods. It plays an important role in the arctic ecosystem and its parasites are transferred to predatory fish, birds and mammals through the food web.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Comparative post-hatching ontogeny of Pseudoplatystoma reticulatum (Eigenmann & Eigenmann 1889) and Leiarius marmoratus (Gill, 1870) and their hybrid

Ontogenetic studies of the eggs and larvae of fish can provide information on the initial life history and biology of a species, are important for taxonomic and evolutionary studies, and for cultivation in captivity. The aim of this study was to analyze and describe the main morphological differences in the larval ontogeny of Pseudoplatystoma reticulatum, Leiarius marmoratus, and its hybrid (♀ P. reticulatum × ♂ L. marmoratus), as well as to identify characteristics that can identify the species and their hybrid at the larvae and juvenile stages. 205 L. marmoratus, 210 P. reticulatum, and 205 hybrid specimens were analyzed, all of which were obtained through induced reproduction. Analyses were performed from hatching to 30 days post-hatching. 19 morphometric and 5 meristic characteristics were evaluated, in addition to chromatophore shape and distribution. The specimens were classified into two life periods: Larval (stages: yolk sac, pre-flexion, flexion, and post-flexion) and Juvenile. Newly hatched larvae were transparent, poorly developed, and had a scarcity of chromatophores. During the early stages of larval development, the three groups showed similarities in appearance and proportional dimensions. However, at both the end of the post-flexion stage and at the juvenile period when individuals were approximately 2 cm long, it was possible to differentiate between hybrids and their parental species by their morphometric, meristic, and pigment characteristics. The hybrid, despite occupying an intermediate position in relationship to its parents, exhibited a shape and size more similar to P. reticulatum, its maternal parent.     

Using molecular genetics to identify immature specimens of the weevil Ceratapion basicorne (Coleoptera: Apionidae)

Molecular analyses can play a primary role in the process of host specificity evaluation at species and population levels. Here we present an example of their application with a promising candidate biological control agent for yellow starthistle, Centaurea solstitialis L. Although it is highly host specific, Ceratapion basicorne (Coleoptera: Apionidae) can develop on safflower in laboratory tests. A field experiment was conducted to further evaluate host plant specificity; however, it was not possible to rear all larvae to the adult stage, which was necessary for species determination. Therefore molecular genetic methods were used to identify immature specimens. A 731 bp fragment of mtDNA cytochrome C oxidase I gene (COI) was sequenced from 41 individuals of C. basicorne and four congeners: Ceratapion orientale, Ceratapion onopordi, Ceratapion penetrans and Ceratapion scalptum. Intraspecific variability ranged from 0.0% to 0.2%, and interspecific divergences ranged from 1.7% to 17.6%. All larvae that were sequenced from the field study, clearly matched one of the five species, enabling us to unambiguously identify them. Use of molecular genetics to identify larvae should also help the process of foreign exploration, enabling the identification of field-collected larvae, which often provide more reliable host plant associations than field collected adults.     

Cloning and Characterization of a Mosquito Larvicidal Toxin Produced During Vegetative Stage of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> 2297

The mosquitocidal toxin 1 (mtx1) gene from genomic DNA of B. sphaericus strain 2297 was cloned and expressed in E. coli. DNA sequencing analysis of the cloned gene revealed a single open reading frame encoding an 870-amino acid polypeptide. Expression level of the full-length gene in E. coli was very low even though strong promoter was used or the gene was expressed as a fusion protein. Expression level was highly improved after the putative leader sequence was deleted, and the truncated gene was expressed as a fusion protein with glutathione S-transferase (GST-tMtx1). E. coli cells expressing GST-tMtx1 was highly toxic to Culex quinquefasciatus larvae and showed lower toxicity against Anopheles dirus and Aedes aegypti larvae. Enterobacter amnigenus An11, a mosquito larval gut colonizable bacteria, transformed with the cloned gene exhibited mosquito larvicidal activity. Result suggested that there is a potential to develop this protein to be used as an alternative mosquito control agent.     

Inferring larval taxonomy and morphology in Maladera species (Coleoptera: Scarabaeidae: Sericini) using DNA taxonomy tools

Based on a comparative molecular study of scarab chafers we matched adult and larval instars to identify and describe unknown larvae of Sericini. Here, we use for the first time a two‐fold DNA taxonomy approach based on: (i) mitochondrial and nuclear DNA markers of a local sample (from Nepal) of adults and larvae, in combination with character and tree‐based species delimitation methods; and (ii) a global search of cytochrome c oxidase subunit I (cox1) sequences with GenBank data. In the latter analysis we used a sequence of a specimen that resulted in the first analysis conspecific with the larvae of Maladera affinis (Blanchard) as the query sequence in GenBank, and checked in a minimum evolution tree whether larva–adult matches from the local approach were altered through interference with other taxa of the worldwide database. Both approaches unambiguously identified the unknown larvae as belonging to M. affinis and Maladera cardoni (Brenske). Based on this robust framework of taxonomic identification we could associate names to the larval morphology of the third larval instar of these two Nepalese Maladera species, which are both known for their economical importance in agriculture. They are described here in detail and are compared with known related taxa, especially with Maladera castanea (Arrow).     

Sex determination in biological specimens using the <Emphasis Type="Italic">DYS14</Emphasis> marker

The applicability of real-time PCR amplification of the chromosome Y marker DYS14 for sex determination was studied. With this aim, real-time PCR of DYS14 (located within the TSPY1-encoding gene) was performed in plasma DNA specimens obtained from 30 men and 30 women. The PCR results showed that 30 specimens were of male and the other 30 were of female origin. All the results were confirmed by the tests for the SRY marker conventionally used in forensic examination. The detection limit for the DYS14-containing DNA region was established in dilution experiments and was equal to 6.7 pg of DNA (two copies of the genome), which corresponds to 6.7 ng of DNA (2000 copies of the genome) in 1 ml of blood. This level of sensitivity allows sex determination in specimens with small amounts of genetic material. The method can be used for noninvasive prenatal diagnostics of sex-linked congenital diseases and in forensic medical examination.     

Molecular Diet Analysis of Phyllosoma Larvae of the Japanese Spiny Lobster <Emphasis Type="Italic">Panulirus japonicus</Emphasis> (Decapoda: Crustacea)

To clarify the natural diet of phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus, the sources of 18S rDNA clones obtained from the hepatopancreas were investigated. Of a total of 1537 clones examined, 160 had different restriction profiles from the host larvae, in which 21 restriction types were observed. Nucleotide sequences of 16 of 21 restriction types were successfully determined and their assignments were investigated by homology search and phylogenetic analysis. From seven late-stage larvae collected in spring to early summer, eukaryote DNA molecules of Teleostei, Oomycetes, Mycetozoa, and Fungi were identified. Exogenous DNA from four younger phyllosoma larvae collected in late autumn could not be recovered. A previous study identified DNAs of cnidarians and urochordates in late-stage phyllosoma larvae of a closely related species collected in winter. This indicates that the phyllosoma larvae are opportunistic carnivores, whose diets correlate with the relative abundance of prey organisms in the ambient water.     

Identification of the ambrosia fungus of <Emphasis Type="Italic">Xyleborus monographus</Emphasis> and <Emphasis Type="Italic">X. dryographus</Emphasis> (Coleoptera: Curculionidae,Scolytinae)

The scolytid ambrosia beetles Xyleborus monographus and X. dryographus were investigated to identify their nutritional ambrosia fungi. The examination of the oral mycetangia of the beetles, the specialized organs for fungal transport, revealed the dominant occurrence of Raffaelea montetyi, a fungus that was also predominant in the beetle tunnels in the immediate vicinity of the feeding larvae. R. montetyi was previously known only as the ambrosia fungus of the platypodid ambrosia beetle, Platypus cylindrus. These beetle species inhabit the same habitat, mainly trunks of oaks in the Western Palaeartic. The possibility of an exchange of the symbiotic fungus between the ambrosia beetles within their common breeding place is discussed. Consequently, the previous hypothesis of a species-specific association of a single ambrosia fungus with a single beetle species is questioned. A phylogenetic analysis based on DNA sequences classified R. montetyi within the Ophiostomatales of the ascomycetes. The investigation of conidiogenesis of R. montetyi by SEM supported this taxonomic placement and showed the development of the conidia by annellidic percurrent proliferation, identical to the conidiogenesis reported for many anamorph states of the Ophiostomatales.     

Parasitism,seasonality, and diversity of trombiculid mites (Trombidiformes: Parasitengona,Trombiculidae) infesting bats (Chiroptera) in Poland

The study aims to ascertain the diversity of trombiculid species associated with Chiroptera in Poland, and for the first time in the case of research on Central European Trombiculidae, we use both DNA and morphology in an integrative taxonomic approach to determine species identities of trombiculids. The research was carried out from 2015 to 2019. In total, 2725 larvae were collected from 300 specimens of bats belonging to 11 species. Deutonymphs were obtained through laboratory rearing of larvae; few larvae and deutonymphs were collected also from bats' daily roosts. The presence of trombiculid larvae on hosts was observed between July and April of the following year, with the highest numbers recorded in autumn, during bat swarming. Male bats were infested more often than females (16.4 vs. 6.6%). The highest infestation rate was recorded for Barbastella barbastellus, Myotis nattereri and Plecotus auritus, and the highest prevalence of chiggers (>?30%) for Myotis bechsteinii and P. auritus. The larvae found on bats occupied the areas with free access to the host’s skin: auricles, tragus, and snout. Morphological identification of specimens to the species level was hindered by the mosaic distribution of diagnostic traits. Morphological analyses indicated the presence of Leptotrombidium russicum and Leptotrombidium spp. in the examined material, whereas molecular analyses additionally suggested three other potential species assigned to the same genus based on the assessed scope of intrageneric variation (ASAP method). We argue that the identification of the parasitic larvae (chiggers) using morphological characters does not address the question of actual species boundaries, which, in turn, affects the inferences about host specificity and host range.

     

Morphological studies of Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) and larval stages of acuariid nematodes parasitic in Larus dominicanus Lichtenstein (Aves: Laridae) from Argentina

Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) is described from Larus dominicanus Lichtenstein (Aves: Laridae) on the Southwest Atlantic coast (38° 42S, 59° 47W). The main character used to distinguish species of Ancyracanthopsis is the morphology of the ptilina. Thus, although the specimens described here have some differences in the morphology and size of the spicules and in the female genitalia, they were referred to A. winegardi because they have a very similar ptilina. This is the first record of a member of Ancyracanthopsis from larid birds and for A. winegardi in the Southwest Atlantic coast. We have also studied acuariid larvae found inhabiting the gizzard alongside adult specimens of A. winegardi. Among those larvae, two morphological groups were clearly distinguished. The first group was characterised by the absence of ptilina and the presence of spicular primordia and rectal cells (third-stage larvae). The second group could be distinguished by the presence of ptilina and partly-developed genitalia (fourth-stage larvae). In order to identify the larvae, a Principal Component Analysis was applied to morphometric data taken from the third-stage larvae. These results and the morphology of the partly-developed ptilina of the fourth-stage larvae indicated that the larval stages found in L. dominicanus appear to belong to Sciadiocara haematopodi, Cremonte, Navone & Etchegoin, 1999.     

Larval sex identification in the paper wasp <Emphasis Type="Italic">Polistes dominulus</Emphasis> (Vespidae,Hymenoptera)

Identifying the sex of larvae is important in social Hymenoptera. Until now for Polistes wasps it has been necessary to genotype larvae at microsatellite loci, and assign their sex based on homozygosity at these loci. In our study on the paper wasp Polistes dominulus we have found morphological differences between larval sexes that can be used for larvae from the 3^rd instar on to easily and cheaply identify larval sex: the external gonopore and the shape and size of larval gonads. The robustness of these indicators was supported by genotype data at four microsatellite loci. Using gonopore and gonad features for sex assignment will assign diploid males as males, unlike techniques based on genetic loci or chromosomes. Received 12 July 2006; revised 4 January 2007; accepted 5 February 2007.