Laboratory-reared Specimens Research Articles

Phenotypic variability in the shield morphology of wild- vs. lab-reared eumalacostracan larvae

Morphological identification of planktic crustacean larvae is required in many scientific contexts, such as ecology or taxonomy. Due to a still low availability of genetic sequences for many ingroups of Eumalacostraca, this task is still more feasible by morphological methods. Our understanding of eumalacostracan larval morphology is challenged by phenotypic variability. We investigated four eumalacostracan ingroups: Galatheidae, Hippoidea, Raninidae and Stomatopoda. Representatives of all four groups develop through spine-bearing planktic larval stages. Incorporating dorsal and lateral shield outlines into three-dimensional shape analysis of the shields, we compare specimens from the wild with laboratory-reared specimens. Using graphical and statistical analysis methods, we find that at least the lateral morphology of the shields of Hippoidea and Raninidae seems to be too strongly dependent on phylogeny to show phenotypic variability with our current sample size, but Hippoidea do show phenotypic variability in their dorsal shield morphology. In Galatheidae and Stomatopoda, a clear difference in shield morphology can be found between wild-caught and laboratory-reared specimens. This difference likely represents phenotypic variability. The exact environmental signals causing this phenotypic variability are still unknown, but some candidates are discussed.

Morphological changes and ontogenetic development of Amblyseius eharai Amitai & Swirski (Acari: Phytoseiidae)

This paper describes the immature and mature characteristics of Amblyseius eharai Amitai & Swirski in detail to determine its morphological ontogeny based on laboratory-reared specimens. The main changes during ontogeny are as follows: the pronotal shield extends to j6 level and opisthosoma is unsclerotized in the larva; the dorsum has two shields in the protonymph, pronotal shield slightly expended comparing to that of the larva, and the opisthonotal shield smooth and weakly sclerotized. The podonotal and opisthonotal shields in the deutonymph are partially fused, and the dorsal shield is complete, covering almost the entire opisthonotum in adults in both sexes. There are nine pairs of dorsal setae in the larva, 14 pairs in the protonymph, and 19 pairs in both deutonymph and adult. In deutonymphs, gender could be distinguished by the number of setae in the area around the anal valves. In protonymphs, all dorsal, hypostomal setae, and the macrosetae of leg IV appear and are consistent with the subsequent stages. The development of morphological traits in the ontogeny of A. eharai may provide some basic knowledge to distinguish it from closely related species.

Electroantennographic Responses of Wild and Laboratory-Reared Females of Xyleborus affinis Eichhoff and Xyleborus ferrugineus (Fabricius) (Coleoptera: Curculionidae: Scolytinae) to Ethanol and Bark Volatiles of Three Host-Plant Species.

Simple SummaryThe ambrosia beetles Xyleborus affinis and Xyleborus ferrugineus are wood borers reported as secondary vectors of pathogenic fungi that cause lethal vascular diseases in mango, cacao, and trees within the laurel family. The use of specific attractants or repellants is one potential method for monitoring or controlling these pests. Chemical ecology studies to develop such tools often use wild or laboratory-reared beetles without first determining whether there are differences in their responses. We compared the antennal olfactory responses of wild and laboratory-reared X. affinis and X. ferrugineus to bark odors of gumbo-limbo (Bursera simaruba), mango (Mangifera indica) and chinini (Persea schiedeana) with different aging times and used GC–MS to analyze the chemical composition of these bark odors. The antennal responses of laboratory-reared and wild females differed in X. affinis and X. ferrugineus when interacting with odors. In addition, both beetle species displayed stronger antennal responses to aged bark odors of gumbo-limbo and chinini, apparently due to changes in volatile emissions over time.Chemical ecology studies on ambrosia beetles are typically conducted with either wild or laboratory-reared specimens. Unlike laboratory-reared insects, important aspects that potentially influence behavioral responses, such as age, physiological state, and prior experience are unknown in wild specimens. In this study, we compared the electroantennographic (EAG) responses of laboratory-reared and wild X. affinis and X. ferrugineus to 70% ethanol and bark odors (host kairomones) of Bursera simaruba, Mangifera indica, and Persea schiedeana aged for 2, 24, and 48 h. Chemical analyses of each odor treatment (bark species x length of aging) were performed to determine their volatilome composition. EAG responses were different between laboratory-reared and wild X. ferrugineus when exposed to ethanol, whereas wild X. affinis exhibited similar EAG responses to the laboratory-reared insects. Ethanol elicited the strongest olfactory responses in both species. Among the bark-odors, the highest responses were triggered by B. simaruba at 48 h in X. affinis, and P. schiedeana at 24 and 48 h in X. ferrugineus. Volatile profiles varied among aged bark samples; 3-carene and limonene were predominant in B. simaruba, whereas α-copaene and α-cubebene were abundant in P. schiedeana. Further studies are needed to determine the biological function of B. simaruba and P. schiedeana terpenes on X. affinis and X. ferrugineus, and their potential application for the development of effective lures.

Balaustium medicagoense Meyer & Ryke, 1959 newly discovered in New Zealand (Acari: Erythraeiddae): descriptions of all life stages

Balaustium medicagoense Meyer & Ryke, 1959 is reported from New Zealand for the first time. Field collected specimens were reared successfully on pollen grains of Typha orientalis in the laboratory. All life stages of this species are described and illustrated from laboratory-reared specimens as well as field-collected specimens.

Consequences of rapid development owing to cohort splitting: just how costly is it to hurry?

In cohort splitting, diverging sub-cohorts may show substantial differences in their growth and developmental rates. Although in the past, causes and adaptive value of cohort splitting were studied in detail, individual-level consequences of cohort splitting are still rather overlooked. Life history theory predicts that considerably increased growth and developmental rates should be traded off against other costly life history traits. However, it is not clear whether one should expect such associations in adaptive developmental plasticity scenarios, because natural selection might have promoted genotypes that mitigate those potential costs of rapid development. To address these contrasting propositions, we assessed life history traits in the wolf spider Pardosa agrestis, both collected from natural habitat and reared in laboratory. We found that some traits are negatively associated with developmental rates in spiders collected from the wild, but these associations were relaxed to a considerable extent in laboratory-reared specimens. In general, we observed no consistent trend for the presence of developmental costs, although some results might suggest higher relative fecundity costs in rapidly developing females. Our study provides a detailed approach to the understanding of individual-level consequences of cohort splitting, and to the associations between key life history traits in adaptive developmental plasticity scenarios.

Ontogenetic changes in the morphology of Eharius chergui (Acari: Phytoseiidae)

The morphological ontogeny of Eharius chergui (Athias-Henriot, 1960) is described in this paper based on laboratory-reared specimens originating from Marrubium vulgare L. (Lamiaceae) collected in North Island, New Zealand. Malformation was observed in the development of some idiosomal setae in some specimens and was suggested to be related to the host plant M. vulgare.

Morphological ontogeny of Amblydromalus limonicus (Acari: Phytoseiidae)

The morphological ontogeny of Amblydromalus limonicus is described in this paper based on laboratory-reared specimens originated from Auckland, New Zealand. Comparisons between A. limonicus and A. lailae from Australia showed that the protonymphs, deutonymphs and adults of these two species are not distinguishable, but unusual differences are present in larvae. The larvae of “A. lailae” by Schicha of Australia were incorrectly associated with other stages of A. lailae and belong to the genus Euseius. The results of this study highlight a neglected area of research in phytoseiid taxonomy: the careful descriptions of immature stages based on reared specimens.

Description of the zoeal stages of Periclimenes aegylios Grippa d'Udekem d'Acoz, 1996 (Crustacea: Decapoda: Palaemonidae) reared in the laboratory.

The eight zoeal stages of the Mediterranean shrimp Periclimenes aegylios are described and illustrated in detail, using laboratory-reared specimens. This study improved the partial and unpublished descriptions of the zoeae of this species. The complete and accurate definition of the morphology of all the stages now allows comparison of the zoeae of P. aegylios with those of other Mediterranean Periclimenes species, as well as the allopatric Atlantic P. sagittifer, from which P. aegylios was separated in 1996.

Cultivable Bacterial Diversity in the Gut of the Chagas Disease Vector Triatoma dimidiata: Identification of Possible Bacterial Candidates for a Paratransgenesis Approach

Since bacterial symbionts play a vital role in the metabolism of hematophagous insect vectors the method known as paratrangenesis, which consists of the use of cultivable insect symbionts to interfere with the transmission of vector-transmitted pathogens has been shown to be effective in certain vector control oriented studies. In Chagas disease research a recent study introduced transgenes through a parastransgenic approach and prevented the development of a vector species for this disease. However this approach requires a previous characterization of the bacterial symbionts present in the species vector of interest, since a selection of the cultivable bacterial symbiont used is mandatory. In this study, we describe the gut bacterial diversity of Triatoma dimidiata specimens from Southern Mexico. Bacteria from both wild and laboratory-reared specimens were cultured, and resulting colonies were grown individually, harvested, and subsequently identified by 16S ribosomal loci sequencing. A total of five and three genera and a total of nine and six bacterial species were identified from field captured and laboratory reared Triatoma dimidiata specimens respectively. A majority of Gram positive bacteria were identified, which included the genera Staphylococcus, Bacillus, Brevibacterium, Micrococcus and Delftia. Given previous studies we propose the use of Staphylococcus saprophyticus, Micrococcus luteus and Bacillus megaterium as potential candidates for future paratransgenic efforts done with Triatoma dimidiata, which is one of the most important vectors of Chagas disease, in Central and South America. Given the vital association bacterial symbionts play in the metabolism of routes of hematophagous insect vectors Paratransgenesis consists of the use of cultivable insect symbionts to interfere with the transmission of vector-transmitted pathogens.

Morphological Description of the Immature Stages of Nyssomyia umbratilis (Ward & Frahia) (Diptera: Psychodidae: Phlebotominae), the Main Vector of Leishmania guyanensis Floch (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon Region.

Nyssomyia umbratilis (Ward & Frahia) is the main vector of Leishmania guyanensis in the Brazilian Amazon region, where it is widely distributed. Studies have hypothesized that this phlebotomine is part of a cryptic species complex, spatially delimited by the Amazonian river systems, and with different transmission potential of L. guyanensis. In the present study, the immature stages of N. umbratilis are described from laboratory-reared specimens originating from the states of Amazonas and Pará, Brazil. Based on scanning electron and optical microscopy examinations, fine morphological structures (eggshell, setae, and mouthparts of larvae and pupae) are presented. A new form of sandfly pupae sexing (non-invasive) is also provided. The correct identification of insect is extremely important for the epidemiology of certain diseases. However, only the morphological characters of adult can be insufficient to separate accurately the closely related species. It is expected that the present description may contribute to solve the taxonomic problem involving N. umbratilis.

Chromatophore arrangement and photophore formation in the early development of swordtip squid Uroteuthis (Photololigo) edulis

The commercially valuable swordtip squid Uroteuthis (Photololigo) edulis has recently experienced a decline, indicating a need for its management. However, very little is known about its morphology during early developmental stages. Therefore, in this report, we describe the chromatophore arrangement and photophore formation in laboratory-reared specimens ranging from 2.2 to 13.6 mm in dorsal mantle length (DML). We observed seven characteristic chromatophore arrangements and a pair of photophores on the ventral surface of the ink sac in the five developmental stages after hatching, which allow U. edulis hatchlings and paralarvae to be distinguished from other sympatric loliginid squids around the coastal area of central Japan. We also found an increase in the number of other chromatophores and rapid growth of the photophores between 7 and 9 mm in DML, which marks the transition between the paralarva and juvenile stages. These findings will be useful for identifying U. edulis hatchlings and paralarvae from natural plankton samples, thus contributing toward research on the spatial distribution, seasonal migration, and predicted spawning grounds of this species.

Effects of experimental Angiostrongylus cantonensis infection on the reproductive biology of Biomphalaria straminea and Biomphalaria tenagophila.

Eosinophilic meningoencephalitis is an endemic zoonosis in Southeast Asia and the Pacific Islands, but in recent years, new cases have been reported in various countries outside these regions, including Brazil, where it is considered an emerging disease. In this study, the effect of infection by the nematode Angiostrongylus cantonensis, one of the main etiologic agent of this disease, on the reproductive biology of the planorbid snails Biomphalaria straminea and B. tenagophila was investigated during the pre-patent period. Alterations in the reproductive biology of B. straminea and B. tenagophila were analyzed in laboratory-reared specimens infected by A. cantonensis during 21days; the number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, viable eggs/snail, survival and galactogen content in the albumen gland were measured. The results indicated the occurrence of initial compensation in reproductive effort in both snail species, but at different moments in the pre-patent period. More specifically, a reduction of 46.53% in the eggs/egg mass ratio in infected B. straminea was observed, a reflection of a 50% decline in the concentration of galactogen contained in the albumen gland. Changes in this parameter were also noted in B. tenagophila, but only at the end of the study period, with a reduction of 15.49%. Histological analyses indicate that changes observed can be explained by the tissue damages caused by the migration and development of the larvae. These results shed more light on the host-parasite relationship and indicate the importance of studying reproductive aspects for efforts to control infected snails. Considering that terrestrial snails can also transmit eosinophilic meningitis (in addition to aquatic mollusks), the data obtained expand knowledge of this host-parasite relationship and provide support for programs to control this zoonosis.

Field application of MALDI-TOF MS on mosquito larvae identification.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an efficient tool for arthropod identification. Its application for field monitoring of adult mosquitoes was demonstrated, but identification of larvae has been limited to laboratory-reared specimens. Study aim was to test the success of MALDI-TOF MS in correctly identifying mosquito larvae collected in the field. Collections were performed at 13 breeding sites in urban areas of Marseille, a city in the South of France. A total of 559 larvae were collected. Of these, 73 were accurately morphologically identified, with confirmation either by molecular identification (n = 31) or analysis with MALDI-TOF MS (n = 31) and 11 were tested using both methods. The larvae identified belonged to six species including Culiseta longiareolata, Culex pipiens pipiens, Culex hortensis, Aedes albopictus, Ochlerotatus caspius and Anopheles maculipennis. A high intra-species reproducibility and inter-species specificity of whole larva MS spectra was obtained and was independent of breeding site. More than 92% of the remaining 486 larvae were identified in blind tests against the MS spectra database. Identification rates were lower for early and pupal stages, which is attributed to lower protein abundance and metamorphosis, respectively. The suitability of MALDI-TOF MS for mosquito larvae identification from the field has been confirmed.

Prolongation of somitogenesis in two anguilliform species, the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus, with refined descriptions of their early development.

The embryonic development of the Japanese eel Anguilla japonica and pike eel Muraenesox cinereus was morphologically investigated with laboratory-reared specimens to clarify the characteristics of somitogenesis. In A. japonica, somites were first observed at 18 h post fertilization (hpf) when epiboly reached 90%. Somitogenesis progressed at a rate of 1·6 h-1 at mean ± s.d. 22·6 ± 0·7° C and completed at 107 hpf (3 days post hatching; dph) when total number of somites (ST) reached 114, which corresponds to the species' number of vertebrae (112-119). In M. cinereus, somites were first observed at 14 hpf when epiboly completed. Somitogenesis progressed at a rate of 1·9 h-1 at mean ± s.d. 24·4 ± 0·2° C and completed at 90 hpf (2 dph) with 149 ± 4 ST, which corresponds to the species' number of vertebrae (142-158). Both species hatched before somitogenesis was completed, at 37 hpf with 47 ST and 42 hpf with 82 ± 4 ST, respectively. The formation of other organs such as the heart, mouth and pectoral fin bud occurred during somitogenesis. Comparison with the development of zebrafish Danio rerio indicates a prolongation of somitogenesis in A. japonica and M. cinereus. Their somitogenesis rates, however, correspond well with that of D. rerio estimated at the same temperature and their developmental stages at hatching are almost equivalent to other fishes having similar yolk sizes. Therefore, the prolongation of somitogenesis in A. japonica and M. cinereus may be accounted for solely by the increased numbers of somites to be formed, not by a slow somitogenesis rate or an acceleration in organogenesis.

SEARCHING FOR NATIVE EGG-PARASITOIDS OF THE INVASIVE ALIEN SPECIES HALYOMORPHA HALYS STÅL (HETEROPTERA PENTATOMIDAE) IN SOUTHERN EUROPE

Halyomorpha halys is an invasive species, originating from East Asia, which was accidentally introduced in some areas of North America and Europe, where severe damages to different vegetable crops and fruit plants were recorded. Several studies in different countries focused on the possibility to apply biological control by means of egg- parasitoids. The main egg-parasitoids in the original area are unfortunately non-specific and thus the application of classical biological control is questionable. On the other hand, the possibility of using augmentative biological control by native egg-parasitoids able to exploit the new host is an interesting possibility in both Europe and the USA. In a preliminary assay, frozen egg masses of H. halys were exposed in the field in Central Italy. Results showed that some eggs were exploited by parasitoids belonging to the species Anastatus bifasciatus and Ooencyrtus telenomicida . Therefore, different native egg-parasitoids species widespread in Europe were tested in the laboratory in order to explore their ability to parasitize H. halys eggs. To this end, no-choice tests were carried out in climatic chambers (26°C, 70%RH, 16:8 L:D) in order to evaluate the parasitization potential of females collected in the wild. Conducted tests pointed out that A. bifasciatus and especially O. telenomicida may be potential candidates for the biological control of H. halys . In particular, O. telenomicida was able to parasitize the 35.56% of the exposed host eggs in the lab tests. New adults successfully emerged from the 22.92% of the eggs, and on the whole, this egg-parasitoid caused a significant hatching reduction of the H. halys eggs (more than 70%). Conversely, Telenomus chloropus and Ooencyrtus pityocampae were less promising species as biocontrol agents of this agricultural pest. Preliminary trials in pear orchards consisting of field releases of laboratory-reared specimens of O. telenomicida, one of the potential candidates for biological control, showed that this parasitoid is actually able to discover egg masses of H. halys (sentinel frozen egg masses) and to successfully parasitize them.

Effect of Cadmium on the Embryonic Development and Hatching Rhythm of Big Fin Squid Sepioteuthis lessoniana, Lesson (1830) from Palk Strait, East Coast of India

In the present study, the impact of cadmium on embryonic development, morphology of eggs and newly hatched larvae of the Sepioteuthis lessoniana were described using the laboratory-reared specimens originating from Thondi, South East coast of India. The eggs were exposed to a concentration of 25 μg Cd/L for up to hatching. The average incubation period of the eggs varied between 18-23 days and the estimated mean temperatures were 18.6 ± 0.7°C for control, and 19.2 ± 1.1°C for Cd exposed groups. The results revealed that a total hatching rate of 99.2 ± 1.6% was achieved in control group and only 65.2 ± 8.8% in treated group. The deformities of the paralarvae were illustrated with light and scanning electron microscopy. In conclusion, the present concentration exhibited toxic effects to the eggs and paralarvae of S. lessoniana. Therefore, Cd level as high as we used in this study could give good information on dangerous Cd levels on squid.

The moulting cycle and changes in body density in larvae of the snow crab Chionoecetes opilio (Brachyura: Majoidea) under laboratory conditions

The moulting cycle and the time course of changes in body density from hatching to the end of the megalopal stage in snow crab (Chionoecetes opilio) larvae were investigated in laboratory-reared specimens. Morphological changes in the epidermis and cuticle were photographically documented to characterize the moult-cycle stages: A–B (postmoult), C (intermoult), D (premoult) and E (ecdysis). Moult-stage characteristics were based on a microscopical examination of integumental modifications, particularly of the telson. During stages A–C, the larval cuticle changed from a spongy structure to become conspicuously thicker and more solid in appearance. In stage D, the epidermis retracted from the cuticle and new setae and appendages were formed. The body densities of larval snow crabs were lowest just after moulting; they increased greatly during stage C, and then gradually increased to reach a plateau at 1.0897–1.0931 g cm−3 during stage D. Over the whole larval period, they have a density greater than that of seawater. These observations will assist in understanding of larval distribution and transport in snow crabs in their natural habitat, and provide a useful tool to determine the developmental stages of larvae sampled from the plankton and from larval cultures.

Accurate identification of Culicidae at aquatic developmental stages by MALDI-TOF MS profiling.

BackgroundThe identification of mosquito vectors is generally based on morphological criteria, but for aquatic stages, morphological characteristics may be missing, leading to incomplete or incorrect identification. The high cost of molecular biology techniques requires the development of an alternative strategy. In the last decade, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has proved to be efficient for arthropod identification at the species level.MethodsTo investigate the usefulness of MALDI-TOF MS for the identification of mosquitoes at aquatic stages, optimizations of sample preparation, diet, body parts and storage conditions were tested. Protein extracts of whole specimens from second larval stage to pupae were selected for the creation of a reference spectra database. The database included a total of 95 laboratory-reared specimens of 6 mosquito species, including Anopheles gambiae (S form), Anopheles coluzzi (M form), Culex pipiens pipiens, Culex pipiens molestus, Aedes aegypti and 2 colonies of Aedes albopictus.ResultsThe present study revealed that whole specimens at aquatic stages produced reproducible and singular spectra according to the mosquito species. Moreover, MS protein profiles appeared weakly affected by the diet provided. Despite the low diversity of some MS profiles, notably for cryptic species, clustering analyses correctly classified all specimens tested at the species level followed by the clustering of early vs. late aquatic developmental stages. Discriminant mass peaks were recorded for the 6 mosquito species analyzed at larval stage 3 and the pupal stage. Querying against the reference spectra database of 149 new specimens at different aquatic stages from the 6 mosquito species revealed that 147 specimens were correctly identified at the species level and that early and late developmental stages were also distinguished.ConclusionsThe present work highlights that MALDI-TOF MS profiling may be useful for the rapid and reliable identification of mosquito species at aquatic stages. With this proteomic tool, it becomes now conceivable to survey mosquito breeding sites prior to the mosquitoes’ emergence and to adapt anti-vectorial measures according to the mosquito fauna detected.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-014-0544-0) contains supplementary material, which is available to authorized users.

Larval and juvenile development of Pike Gudgeon, Pseudogobio esocinus (Cyprinidae: Gobioninae)

We describe the larval and juvenile development of Pike Gudgeon, Pseudogobio esocinus, using laboratory-reared specimens. Newly hatched larvae with a 4.2–4.6 mm body length (BL) and 22–23 + 16 = 38–39 myomeres had melanophores on their head and body. The yolk sac was completely absorbed at 5.0 mm BL. Notochord flexion was initiated at 5.0–6.0 mm BL and completed at 7.3 mm BL. The aggregate number of all fin rays was completed at 11 mm BL. Several rod-like cupulae were observed on the head and lateral side of the body at 4.2–11.0 mm BL and were completely distinguished at 11.0–14.0 mm BL. Squamation was initiated on the caudal body at approximately 11.0 mm BL and completed at 11.0–14.0 mm BL. The newly hatched larvae had well-developed eyes and large pectoral fins.

Hylid Tadpoles from the Caribbean Island of Hispaniola: Ontogeny, Description and Comparison of External Morphology

Abstract The island of Hispaniola (Dominican Republic and Haiti) is home to four endemic species of treefrogs: Hypsiboas heilprini, Osteopilus dominicensis, O. pulchrilineatus and O. vastus. These species occur sympatrically over large areas below 1,850 m elevation and are commonly encountered together. We redescribe the external larval morphology of O. dominicensis, O. vastus, and Hybsiboas heilprini, and describe the tadpole of O. pulchrilineatus for the first time based on laboratory-reared specimens representing stages from early—late metamorphosis. Some tadpoles were raised to adults in order to confirm species identity. We document intraspecific variation in labial tooth-rows formulas, report ontogenetic changes in coloration, compare tadpoles of related species, and provide an identification key to the hylid tadpoles of Hispaniola. Hispaniolan hylid tadpoles inhabit fast-running streams or pools. Whereas the lotic larvae of O. vastus and H. heilprini exhibit characteristics commonly associated with...