Pathology of black bass hepatic tissue infected with larvae of the tapeworm Proteocephalm ambloplitis

Intra- and interspecific differences in pigmentation between finfold larvae of the three most abundant cyprinids in Dutch eutrophic waters, bream, white bream and roach, were studied, using laboratory-raised larvae in the length range 8–11 mm. Because the internal pigmentation of the larvae has been used for identification, some attention is paid to the effects of different ways of fixation and preservation on transparency. The size and the shape of the melanophores, as described in the literature, could not be used as identification characters because of too much intraspecific variation and the effects of light conditions at the moment of fixation. Three characters proved to be significant for the identification of the larvae of the three species. Roach can be distinguished from bream and white bream by the pattern of melanophores on the belly. A second character is the pigmentation of the ventral aorta, which is only found in white bream. Lastly bream shows an irregular pattern of melanophores on the dorsal side, in contrast to roach which has a regular pattern.     

Noninvasive molecular methods to identify live scarab larvae: an example of sympatric pest and nonpest species in New Zealand

Despite the negative impact that many scarab larvae have on agro-ecosystems, very little attention has been paid to their taxonomy. Their often extremely similar morphological characteristics have probably contributed to this impediment, which has also meant that they are very difficult to identify in the field. Molecular methods can overcome this challenge and are particularly useful for the identification of larvae to enable management of pest species occurring sympatrically with nonpest species. However, the invasive collection of DNA samples for such molecular methods is not compatible with subsequent behavioural, developmental or fitness studies. Two noninvasive DNA sampling and DNA analysis methods suitable for the identification of larvae from closely related scarab species were developed here. Using the frass and larval exuviae as sources of DNA, field-collected larvae of Costelytra zealandica (White) and Costelytra brunneum (Broun) (Scarabaeidae: Melolonthinae) were identified by multiplex PCR based on the difference in size of the resulting PCR products. This study also showed that small quantities of frass can be used reliably even 7 days after excretion. This stability of the DNA is of major importance in ecological studies where timeframes rarely allow daily monitoring. The approach developed here is readily transferable to the study of any holometabolous insect species for which morphological identification of larval stages is difficult.     

On the Roles of Morphological and Biochemical Criteria in Species Identification: An Example of Dragonfly Larvae of the Genus Aeschna

Dragonflies belong to the group of organisms with numerous well-differentiated species-specific characters at the adult stage, on the one hand, and a significantly smaller number or even the absence of such characters at the early ontogenetic stages. An example of the genus Aeschna is used to show difficulties in revealing morphological and biochemical characters allowing identification of larval dragonflies belonging to closely related species of the family. Distinct morphometric characters can be found only in late-instar larvae. The presence of species-specific proteins in the homogenates of thoracic muscles provides the possibility of using biochemical tests for species identification of larvae. Infestation by parasites has no effects on the biochemical parameters studied. Species identification of the early-instar dragonfly larvae is still problematic.     

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5'-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.     

A Novel Technique for Identifying the Instar of Field-Collected Insect Larvae

Many field studies of insects have focused on the adult stage alone, likely because immature stages are unknown in most insect species. Molecular species identification (e.g., DNA barcoding) has helped ascertain the immature stages of many insects, but larval developmental stages (instars) cannot be identified. The identification of the growth stages of collected individuals is indispensable from both ecological and taxonomic perspectives. Using a larval–adult body size relationship across species, I present a novel technique for identifying the instar of field-collected insect larvae that are identified by molecular species identification technique. This method is based on the assumption that classification functions derived from discriminant analyses, performed with larval instar as a response variable and adult and larval body sizes as explanatory variables, can be used to determine the instar of a given larval specimen that was not included in the original data set, even at the species level. This size relationship has been demonstrated in larval instars for many insects (Dyar’s rule), but no attempt has been made to include the adult stage. Analysis of a test data set derived from the beetle family Carabidae (Coleoptera) showed that classification functions obtained from data sets derived from related species had a correct classification rate of 81–100%. Given that no reliable method has been established to identify the instar of field-collected insect larvae, these values may have sufficient accuracy as an analytical method for field-collected samples. The chief advantage of this technique is that the instar can be identified even when only one specimen is available per species if classification functions are determined for groups to which the focal species belongs. Similar classification functions should be created for other insect groups. By using those functions together with molecular species identification, future studies could include larval stages as well as adults.     

Differentiation of subspecies of the polymorphic species Hyalomma marginatum (Acari: Ixodidae) based on immature stages

Study of morphological characters to identify the subspecies of Hyalomma marginatum immature stages is based on material collected throughout all the geographical range of the species. As it was found, the discrimination of subspecies of H. marginatum immature stages needs a complex use of structural and morphometrical characters. Only H. m. marginatum larvae and H. m. isaaci nymphs may be easily differentiated from other subspecies based on the structural (qualitative) characters. Besides, morphometric (quantitative) characters should be used only in combinations for identification of the subspecies. In regard to structural characters of larvae, it was found that the shape and size of coxal spurs of H. marginatum allow differentiating this subspecies from the others (Fig. 3, 5, 6). Based on morphometrical characters of larvae, the following characteristic features of subspecies have been found: in H. m. marginatum, the capitulum is wide, the palpi, hypostome and genua I are short and narrow; in H. m. turanicum, the capitulum is narrow, the palpi are short and narrow, the hypostome is short and wide, the genua I are long and narrow; in H. m. rufipes, the scutum is large, the capitulum is wide, the palpi are long and narrow, the hypostome and genua I are long and wide; in H. m. isaaci, the scutum is small, the capitulum is narrow, the palpi and hypostome are long and narrow, the genua I are short and narrow. Among structural characters of nymph, several discriminative features of subspecies have been found. The shape of the scutum in H. m. isaaci (Fig. 2, 2) clearly differentiates this subspecies from the others (Fig. 2, 1). In H. m. marginatum, the setae of alloscutum as a rule have bluntly rounded apices (Fig. 1, 2), while in the other subspecies these setae are more tapering apically (Fig. 1, 1). The shape of spiracular plates is rather variable within the species, but in H. m. marginatum and H. m. isaaci (Fig. 1, 3) the plates are larger and more perforated than in H. m. rufipes and H. m. turanicum (Fig. 1, 4). In H. m. isaaci, the spurs of coxae I are narrower than in other subspecies (Fig. 1, 8). Based on morphometrical characters of nymphs, the following discriminative features of subspecies have been found: in H. m. marginatum, the capitulum is wide, the palpi are short and wide; in H. m. turanicum, the capitulum is narrow, the palpi are long and narrow; in H. m. rufipes, the scutum is wide, the capitulum is narrow, the palpi are long and narrow, the hypostome is long; in H. m. isaaci, the scutum is long and narrow, the capitulum is wide, the palpi are short and wide, the hypostome is short.     

Phylogenetic analysis of the genus <Emphasis Type="Italic">Argia</Emphasis> Rambur, 1842 (Odonata: Coenagrionidae), based on morphological characters of larvae and mitochondrial DNA sequences

The study of the evolutionary interrelationships among the species encompassed in the Neotropical genus Argia (Zygoptera: Coenagrionidae) has been neglected. The goal of this study is to infer the phylogenetic relationships among 36 species of Argia Rambur, 1842, using complementary data sets (i.e., larval morphology and mitochondrial DNA). The morphological data set comprises 76% of the larvae currently described for this genus and includes 97 morphological characters. From those, 47 characters have not been previously used in taxonomic studies involving dragonflies’ larvae. This is the first cladistic study based on larvae morphology for species within the suborder Zygoptera. Data partitions were analyzed individually, as well as total evidence, using parsimony and Bayesian inference as criteria for optimal-tree selection. The results support the monophyly of the North American species of Argia. This genus can be identified by the combination of eight synapomorphies, four of which are exclusively found in Argia. According to the optimal trees, the individual data sets (i.e., morphology and DNA sequences) have a high level of homoplasy, resulting in soft polytomies and low support for several nodes. The specific relationships of the terminal units differ between the phylogenies; nonetheless, there is historical congruence among them. Within Argia, five clades were consistently recovered. Most of those clades have been identified, at least in part, in previous phylogenetic and taxonomic studies. Indubitably, the morphological characters from larvae have historical signal useful for cladistic and taxonomic inference. Therefore, it should be a priority to pay more attention to this source of characters.     

Olfactory responses of Grapholita molesta to different developmental stages of immature fruit of peach,plum, and apple

The Oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae), is known to shift its host use from stone fruits (the primary hosts) to pome fruits (secondary hosts) as the season progresses. Grapholita molesta recognizes potential hosts by semiochemicals from fruits, but few studies have addressed the olfactory responses of G. molesta to host fruits during the seasonal transition of the moth from stone to pome fruits. So, we investigated the olfactory responses of larvae and gravid females of G. molesta to different fruit stages (collected on May 31, June 11, and June 25) of immature peach, plum, and apple fruits, using both multiple and two choice tests. In the multiple choice tests, for fruits collected after June 11, larvae showed a significant preference for peach as their first choice, compared to plum or apple. The final choice rate (when larvae had stayed on the fruit for more than 1 h) of larvae was also highest for peach fruits collected on June 11 and June 25. In two choice tests (Y-tube test), the response rate and overall choice rate of larvae and mated ovipositing adult females were also highest on peaches collected on May 31 (larvae) and June 25 (adults), respectively. In conclusion, we found that both the adults and larvae of G. molesta were highly attracted to immature peach fruits in laboratory choice tests.     

Vegetative Identification of Tropical Woody Plants: State of the Art and Annotated Bibliography1

This annotated bibliography is provided in order to assess the achievements and gaps in the literature on vegetative identification of woody plants in the tropics. The bibliography includes 258 references divided into general references (48), pantropical and multi‐continental keys (4), Africa (46), Asia (53), Australia and New Guinea (21), and the Neotropics (86). Vegetative keys are not easy shortcuts to the identification of woody tropical plants to species. Identification of sterile material requires as much, if not more, training as that required for using keys that emphasize floral and fruit characters. Nevertheless, the literature surveyed in this bibliography may be helpful to biologists seeking references for the geographic region in which they work, models for the construction of vegetative keys and field guides, and/or the types of characters that should be observed in the field.     

Using in situ hybridization to expand the daily egg production method to new fish species

The capacity to reliably identify fish eggs is critical in the application of the daily egg production method (DEPM) to estimate biomass of commercially important species. This application has largely been confined to species that have easily identifiable eggs. Various molecular strategies have been used to extend the DEPM to a broader range of species, with recent approaches like in situ hybridization (ISH) that preserves the integrity of whole eggs, embryos or larvae recommended as a suitable alternative over destructive procedures like PCR. Here, we designed and validated an ISH approach for the identification of whole eggs and larvae from Snapper (Chrysophrys auratus) from environmental samples using the mitochondrial 16S rRNA gene as a target for specific horseradish peroxidase (HRP)‐conjugated oligonucleotide probes. This colorimetric assay allowed the highly specific detection of positive hybridization signals from intact C. auratus larvae and eggs from mixed‐species samples comprising closely related taxa. Furthermore, evaluation of whole eggs across a range of developmental stages revealed the sensitivity of the approach for discerning early stages, thereby guiding staging and the identification of otherwise indistinguishable eggs from environmental samples. This approach represents a major advance from current molecular‐based strategies as it is nondestructive and allows for the simultaneous identification and staging of fish eggs (and larvae). The resultant 100% egg identification certainty we have achieved allows the DEPM to be applied to a wider array of fish species and is particularly applicable to species in areas where morphologically similar eggs are being spawned at the same time.     

Indiscriminate oophagy by ant larvae: an explanation for brood serial organization?

Summary Last instar larvae ofLasius niger under standard laboratory conditions and abundant food supply feed on conspecific eggs and, if forced to a choice, show a significant statistical preference for trophic versus fertilized eggs (approx. 60% of the trials observed). On the other hand, they are unable to discriminate between kin and non-kin eggs, both fertilized and trophic. Fertilized eggs killed by freezing and trophic eggs handled in the same way are also selected in a random manner.Last instar larvae ofMessor semirufus regularly failed to discriminate in a significant way between trophic and fertilized and between kin and non-kin eggs.Substantial experimental evidence confirms that larvae of both these species — as already suggested in the literature for other ants-rely on trophic eggs as an essential component of their diet in order to be able to develop. Additionally,L. niger last instar larvae appear to be able to attack and pierce the egg chorion with their own mandibles, while this capacity is at least very reduced or very rare inM. semirufus. First and second instar larvae of both species never succeeded in piercing the egg membrane alone.These findings imply that the right (i.e. trophic) eggs should be presented to the larvae by the workers (i.e. worker discrimination should be assumed) and, at least in the case of youngLasius larvae, and probably for all stages ofMessor larvae, the egg membrane must be pierced by the workers in order to allow the larvae to feed.The following evolutionary sequence is suggested to explain the origin of trophic eggs in ants: 1) larval oophagy (obligatory at least for the species founding new colonies in an independent claustral manner), 2) generalized facultative or obligatory larval oophagy for larvae of all colonial stages, 3) production of trophic eggs by the queen(s) and/or workers in order to avoid cannibalism of nestmates.Lack of discrimination or weak discrimination capacity between trophic and fertilized eggs and the consequent larval cannibalism in form of oophagy, as demonstrated in this paper, is suggested to explain age segregation among eggs and larvae from workers, a widespread phenomenon in ants which must have been selected to avoid the oophagy of viable eggs.     

On the complementarity of DNA barcoding and morphology to distinguish benign endemic insects from possible pests: the case of Dirioxa pornia and the tribe Acanthonevrini (Diptera: Tephritidae: Phytalmiinae) in Australia

Fruit flies are considered economically important insects due to some species being agricultural pests. However, morphological identification of fruit fly adults and larvae can be difficult requiring a high level of taxonomic expertise, with misidentifications causing problematic false-positive/negative results. While destructive molecular techniques can assist with the identification process, these often cannot be applied where it is mandatory to retain a voucher reference specimen. In this work, we non-destructively (and partial-destructively) processed larvae and adults mostly belonging to the species Dirioxa pornia (Walker, 1849), of the poorly studied nonpest fruit fly tribe Acanthonevrini (Tephritidae) from Australia, to enable molecular identifications whilst retaining morphological vouchers. By retaining the morphological features of specimens, we confirmed useful characters for genus/species-level identification, contributing to improved accuracy for future diagnostics using both molecular and morphological approaches. We provide DNA barcode information for three species of Acanthonevrini known from Australia, which prior to our study was only available for a single species, D. pornia. Our specimen examinations provide new distribution records for three nonpest species: Acanthonevroides variegatus Permkam and Hancock, 1995 in South Australia, Acanthonevroides basalis (Walker, 1853) and D. pornia in Victoria, Australia; as well as new host plant records for D. pornia, from kangaroo apple, apricot and loquat.     

The affinities of mites and ticks: a review

Mites and ticks can be divided into two well-defined clades, Anactinotrichida and Actinotrichida, for which a recent work formalized a suite of putative autapomorphies and reciprocal differences. Whether they are sister-taxa – forming a monophyletic Acari – is more controversial. Earlier supporters of two independent origins for mites largely failed to demonstrate convincing synapomorphies between either of the two lineages and other arachnid orders; although recent work on reproductive biology revealed explicit characters of this nature. Furthermore, some of the characters proposed in support of a monophyletic Acari do not stand up to detailed scrutiny when compared with Arachnida in general. Effective morphological comparisons between mites and other arachnids are hindered by incompatible nomenclature and long-standing, mite-specific characters which are difficult to score for other arachnids. Furthermore, taxon-specific characters restricted to individual mite groups have sometimes been treated erroneously as 'typical' for all Acari. Here, previous hypotheses of mite affinities are reviewed. Historically, authors have debated whether mites are basal arachnids or highly derived. Excluding weakly supported early hypotheses, mites have been resolved – in whole or in part – as sister-group of all other Arachnida (based on tagmosis), closely related to Opiliones (based mostly on genital morphology), Palpigradi (based on controversial interpretations of limb morphology), Solifugae (based mostly on the mouthparts, but now perhaps also reproductive characters) and Ricinulei (based on hexapodal larvae and perhaps mouthparts). We cannot provide a final resolution here, but we aim to highlight important character sets which should be included in subsequent phylogenetic analyses, as well as useful areas for future investigations: particularly tagmosis and the nature of the gnathosoma.     

PromoterExplorer: an effective promoter identification method based on the AdaBoost algorithm

MOTIVATION: Promoter prediction is important for the analysis of gene regulations. Although a number of promoter prediction algorithms have been reported in literature, significant improvement in prediction accuracy remains a challenge. In this paper, an effective promoter identification algorithm, which is called PromoterExplorer, is proposed. In our approach, we analyze the different roles of various features, that is, local distribution of pentamers, positional CpG island features and digitized DNA sequence, and then combine them to build a high-dimensional input vector. A cascade AdaBoost-based learning procedure is adopted to select the most 'informative' or 'discriminating' features to build a sequence of weak classifiers, which are combined to form a strong classifier so as to achieve a better performance. The cascade structure used for identification can also reduce the false positive. RESULTS: PromoterExplorer is tested based on large-scale DNA sequences from different databases, including the EPD, DBTSS, GenBank and human chromosome 22. Experimental results show that consistent and promising performance can be achieved.     

The phylogenetic relevance of thoracic musculature: a case study including a description of the thorax anatomy of Zygoptera (Insecta: Odonata) larvae

New morphological techniques allow for the evaluation of novel character systems that are potentially important for phylogenetic analysis. Only a few studies so far have used character systems from the insect thorax for phylogenetics; the reasons for this might include a lack of common terminology or established homology for pterygote insect thorax musculature. Still, recent studies have proposed common terminology and hypotheses of homology, now allowing for an evaluation of thoracic morphological character systems among the groups of winged insects. Using X ‐ray microtomography (μCT) we present a detailed study of the thorax musculature of O donata as an important phylogenetic character system, with a matrix of 298 characters with 697 character states, including novel data from the thoracic anatomy of eight damselfly larvae. We also included additional O donata, E phemeroptera and N eoptera taxa from the literature and demonstrate the phylogenetic relevance of this character system by reproducing phylogenetic topologies of established relationships. We also compared high‐resolution data from O donata larvae from our study and from recent literature with data from older literature in the adult O donata. All major clades were successfully recovered, (e.g. O donata, E piprocta, A nisoptera and Z ygoptera) with high node support, but obtained higher phylogenetic resolution with the larval data. The best phylogenetic resolution was achieved by combining the adult and larval characters. The taxon sampling and character matrix is the largest to date and underlines the potential relevance of the thorax musculature as an important phylogenetic character system.