Ultramorphological characteristics of immature stages of Chrysomya albiceps (Wiedemann 1819) (Diptera: Calliphoridae), a fly specie of forensic importance

Forensic entomology is an area of science that serves as a tool in crime scene investigations. Usually, flies are the first insects to reach a dead body and can oviposit just a few hours after arrival. Therefore the knowledge of immature stages is essential for correct identification of the species found on corpses. Scanning electron microscopy gives detailed information about morphological characters helping to identify the immature forms and consequently serves as a tool in crime scene investigations. C. albiceps is a very important fly for forensic entomologists because its larvae are almost always present on a dead body and it is facultative predators and therefore can alter the composition of species present at the carcass. The aim of this study is to identify eggs, larvae, and puparia of C. albiceps using SEM. Eggs were elongated with the anterior region ending in a “Y” shape and the posterior end was tapered. The micropyle was a well‐adorned orifice with some projections around it. The first instar larva was composed of 12 segments separated by spines. Only one spiracular opening could be seen at the posterior spiracle. Body tegument was smooth and tubercles were not seen. Antennae and maxillary palps were visible. Second and third larval instars were very similar to first instar, except for the presence of anterior spiracle. However, body tegument was composed of net‐like patches and tubercles were visible. Tubercles present at the third instar larvae were robust and erect. Puparia showed a retracted cephalic region and curved tubercles. Microsc. Res. Tech. 73:779–784, 2010. © 2010 Wiley‐Liss, Inc.     

Morphology of immature stages of Megaselia spiracularis Schmitz (Diptera: Phoridae)

In addition to causing myiasis in humans, Megaselia spiracularis Schmitz has also been reported as a forensically important fly. In this study, we presented the morphology of all larval instars and puparium of M. spiracularis using scanning electron microscopy. The first instar larva was composed of 12 segments. Antennae and maxillary palp complex were visible. Two spiracular slits could be seen at the posterior spiracle. The branch of posterior spiracular hairs was approximately equal to the palm‐formed base in length. Second and third larval instars were very similar to first instar, except for the presence of anterior spiracle. The labium of the second instar larva was triangular and ventrally curved, whereas it was a bilobed structure and the tip forked in the first instar. The bubble membrance comprised of ≈40 globules presented at the third instar larvae. Puparia showed a retracted cephalic region and a pair of pupal respiratory horns on the dorsum. A comparison of the morphological features between immature stages of M. spiracularis and M. scalaris, a forensically important fly indoors in Germany, Malaysia, and China, was discussed. Microsc. Res. Tech. 75:1297–1303, 2012. © 2012 Wiley Periodicals, Inc.     

Morphology of the fertilizable mature egg in the Acanthopagrus latus,A. schlegeli and Sparus sarba (Teleostei: perciformes: sparidae)

Through appropriate scanning electron microscope (SEM) processing, the shape and the surface structures of just‐mature eggs are preserved integrally for observation and measuring to guarantee accurate comparison and prediction on the phylogenetic relationships among three species of teleost, i.e. Acanthopagrus latus, Acanthopagrus schlegeli and Sparus sarba of Sparidae. Eggs in all three species can be categorized as type III eggs. Specifically, a regular pattern is found in the arrangement of micropyle and its surrounding pore canals from the inspected surface structures of the mature eggs in three species. Other than these similarities in egg surface structures, several differences in micropyle structures, micropyle region and size of pore canals are also observed in eggs of respective species. Comprehension and accurate data are statistically computed and analyzed through hierarchical cluster analysis to provide solid species‐specific evidences and standardized empirical reference for accurate species identification and phylogeny determination.     

Identification of Hypoderma actaeon (Diptera: Oestridae) in red deer (Cervus elaphus) from northern Spain: Microscopy study and molecular analysis

Hypoderma spp. larvae were observed subcutaneously in the dorsal and lumbar regions of red deer (Cervus elaphus) hunted in the province of León (northwestern Spain) causing a myiasis. They were removed and initially classified by their size, shape, color, and location under the skin into the three larval stages that parasitize these animals. The morphological characteristics of the first and second-instar are described and from the features of the third-instar the species was identified as Hypoderma actaeon. To accurately identify this species, five isolates of genomic DNA from the third-instar, two from the second-instar and two from first-instar of H. actaeon were analyzed by PCR analysis of the COI region of mt-DNA. The results confirmed that the examined samples exactly matched with H. actaeon. This study has shown the morphological identification of the three larval stages of H. actaeon and, for the first time, the first and second-instar larvae have been molecularly characterized. Finally, identification of only H. actaeon suggests that this species is the only affecting red deer in the Iberian Peninsula.     

Pollen morphology of Chinese Pseudostellaria (Caryophyllaceae) and its systematic significance

Using light and scanning electron microscopy, we studied the pollen morphology of six species (one of which are endemic) of Chinese Pseudostellaria. All species were studied for the first time. Results of this study indicated that pollen grains of Pseudostellaria are spheroidal or spheroidal‐polyhedral in shape and small or medium in size and pantoporate. Each pollen grain has 12–16 round pores. These pores are apart from each other by 5.77–7.73 μm and each has 7–13 granules in the operculum region. These features have important taxonomic significance. In addition, their pollen grains have thin exine with microechinate‐punctate‐perforate surface ornamentation. Pollen features do not support the traditional infrageneric classification of Pseudostellaria into two sections, but support that Pseudostellaria is closely related to other species of the Odontostemma clade.     

Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots

Artificial conditions of tissue culture affect growth and physiology of crassulacean acid metabolism plants which often results in formation of hyperhydric shoots. In in vitro conditions Mammillaria gracilis Pfeiff. (Cactaceae) growth switches from organized to unorganized way, producing a habituated organogenic callus which simultaneously regenerates morphologically normal as well as altered hyperhydric shoots. In this study, influence of tissue culture conditions on morphology of cactus spines of normal and hyperhydric shoots was investigated. Spines of pot‐grown Mammillaria plants and of in vitro regenerated shoots were examined with stereo microscope and scanning electron microscope. The pot‐grown plants had 16–17 spines per areole. In vitro grown normal shoots, even though they kept typical shoot morphology, had lower number of spines (11–12) and altered spine morphology. This difference was even more pronounced in spine number (six to seven) and morphology of the hyperhydric shoots. Scanning electron microscopy analysis revealed remarkable differences in micromorphology of spine surface between pot‐grown and in vitro grown shoots. Spines of in vitro grown normal shoots showed numerous long trichomes, which were more elongated on spines of the hyperhydric shoots; the corresponding structures on spine surface of pot‐grown plants were noticed only as small protrusions. Scanning electron microscopy morphometric studies showed that the spines of pot‐grown plants were significantly longer compared to the spines of shoots grown in tissue culture. Moreover, transverse section shape varies from elliptical in pot‐grown plants to circular in normal and hyperhydric shoots grown in vitro. Cluster and correspondence analyses performed on the scanning electron microscope obtained results suggest great variability among spines of pot‐grown plants. Spines of in vitro grown normal and hyperhydric shoots showed low level of morphological variation among themselves despite the significant difference in shoot morphology.     

Histomicroscopy and normal anatomy of the adult killifish Aphanius hormuzensis (Teleostei; Aphaniidae) from the Persian Gulf coastal environment

The histomicroscopy and normal anatomy of the major body organ systems were investigated in the adult killifish, Aphanius hormuzensis using histological examination, X‐ray imaging, double staining, light microscopy and scanning electron microscopy (SEM). Based on the histomicroscopic observations, the kidney, liver and swim bladder in the studied species were comparable to other fish models. The anterior portion of the kidney is bulbous, while the posterior portion is narrow and elongated; the liver has a single lobe and the swim bladder is a single‐chambered organ with no connection to the digestive tract (physoclistous). X‐ray imaging and double staining examination showed 12 abdominal and 15 caudal vertebrae and a single hypural plate in the caudal skeleton. According to light microscopy, the scales were rounded to pentagonal in shape with three types of radii (primary, secondary and tertiary), and the urohyal bone was elongated. SEM microscopy showed a single row of tricuspid teeth on the upper and lower jaw, respectively, each tooth has two lateral cusps that are shorter than the middle one. The number of teeth was 17–18 in the upper jaw and 19–20 in the lower jaw. The saccular otoliths were rounded‐trapezoid in shape with a moderately incised and V‐shaped excisura. The members of killifishes are an important group for biologists because of their evolutionary properties, regeneration capacity and usefulness as biological control and also for the ecotoxicological assessment of environmental pollution. The outcomes of this study may provide a useful basis for future research on the genus Aphanius.     

Pollen micromorphological analysis of tribe Acacieae (Mimosaceae) with LM and SEM techniques

The morphology of pollen grains of the 06 species of tribe Acacieae (Mimosaceae) belonging to one genus were investigated using scanning electron microscopic (SEM) and light microscopic (LM) approach. Lactic acid acetolysis method was used for (LM) while non‐acetolysis for (SEM). The species were examined for the both qualitative and quantitative palynological features. Qualitatively the pollen was studied for the shape, exine sculpturing, pollen shape, and pollen type. Oblate spheroidal shape of pollen was observed in Accacia modesta. Subprolate shape in Accaca nilotica. In Accacia farnesiana, prolate pollen was examined while prolate spheroidal in Acacia tortilis and spheroidal pollen grains were studied in Acacia catechu, respectively. Pollen exine sculpturing of the studied tribe possesses novel variation. Foveolate, reticulate, foveolate‐psilate, faint reticulate, fossulate, perforate, and scrabate exine sculpturing were noticed. Colpi were absent in all species. Excessive variation was observed in both qualitative and quantitative character of pollen. SPSS software was use for the quantitative measurement of the pollen grains. The current study will be helpful for the identification of the problematic species and phylogenetic studies of family Mimosaceae.     

Description of the immatures of workers of the ant Linepithema micans forel (Hymenoptera: Formicidae)

Linepithema micans Forel is a poorly studied species that is now considered a pest in vineyards in South Brazil. Larval characters have been used in a few phylogenetic studies, and their importance greatly depends in the amount of available information on different species. This study presents a complete panorama on the external morphology of the immatures of L. micans based on observations by light and scanning electron microscopy. The number of larval instars was estimated as three based on the frequency distribution of head widths of 965 larvae. Larvae of L. micans were similar to other Linepithema Mayr in the general format of body and mandibles, presenting a dorsal abdominal protuberance, nine pairs of spiracle, and unbranched hairs. On the other hand, L. micans was unique for having shorter hairs, predominantly denticulate, intraspecific variation in the number of antennal sensilla and in the types of sensilla on the labial palps were reported. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Morphological characterization of Hypnaceae (Bryopsida,Hypnales): Investigating four genera from Western Himalayas by using LM and SEM techniques

The Hypnaceae is one of the largest and diversified family among the pleurocarpous mosses which consists of nearly 60 genera and 1,000 species around the world. In Pakistan, it is represented by 15 species and 8 genera. The current research article provides the detail morphological investigation of four different species (genera) of the family Hypnaceae that is, Gollania clarescens, Hypnum revolutum, Homomallium andoi, and Taxiphyllum taxirameum in the Western Himalayas, Pakistan. The research article reports the first SEM study of the leaf surfaces of the studied taxa, along with comprehensive morphological characters of the four species. The main objective of the research project is to present the comparative light and scanning electron microscopic study to discuss the morphology in detail because previously the family is just reported in different bryophyte checklists of the Western Himalayas. Based on results, morphological characters, micromorphological observations, qualitative and quantitative attributes, taxonomic keys for the studied taxa, distribution, and habitat are described. Stereoscope was used to analyze different qualitative characters, and light microscope was used to observe and measure the laminal and alar cells of the leaf. The SEM study reveals many important surface features like cell shape and cell wall. The laminal cells were mostly linear and elongated with thin cell walls. The specimens used for the SEM were air dried, so the laminal cells were somewhat constricted and concaved. The current study project will help to make the contribution in the taxonomy and morphology of this family.     

Morphology of the third instar larva of Colobopterus quadratus (Coleoptera: Scarabaeidae: Aphodiinae) using scanning electron microscopy

Aphodiinae are a large cosmopolitan subfamily of Scarab beetles (Scarabaeoidea: Scarabaeidae), playing an important role during dung decomposition. Many aspects, especially immature stages, of these dung beetles have not yet been recorded due to their comparative small body sizes and lacking of striking rolling behavior. In this study, the third instar larva of Colobopterus quadratus is morphologically described using scanning electron microscopy. The larva is atypical for the combination of following morphological features: (a) the larval antennae are five‐segmented; (b) the clypeus bears 4 minute protuberances on dorsal surface; (c) the epitorma on epipharynx is ventrally protuberant; (d) the scissorial area of left mandible is broad and blade‐like; (e) the raster is furnished with six glabrous protuberances and two rows of pali arranged in V‐shape. Some significant ultra‐morphological features were discovered in larval Aphodiinae for the first time. The presence of blade‐like incisor on the left mandible implies the potential root cutting ability.     

Characterization of Albanian water frog,Pelophylax shqipericus,sperm traits and morphology,by using phase contrast microscopy

Mature spermatozoa traits and morphology of endangered Albanian water frog, Pelophylax shqipericus, have been characterized for the first time through phase contrast microscopy, as part of successful implementation of in vitro fertilization technique for this species. The basic morphology of P. shqipericus spermatozoa consists of an elongated, thick, smooth‐edged, and solid‐staining head, continuing with a thin and long tail which usually extends 2.48 times the head length. The acrosome was not clearly discernible so the measurements were done on the head as a whole, while the middle section was better visible. Average length of head, including the acrosome and midsection was estimated to be 11.78 μm ± 0.32, while the tail length resulted 29.24 ± 1.75 μm. The average thickness of the head was shown to be 3.45 μm. The total sperm length resulted to be 41.02 ± 1.83 μm. The average sperm concentration was estimated of 25.5 × 10⁶/ml. Sperm amount, survival rate and motility were also measured. The sperm survival rate was maximal immediately after preparation of the suspension and tended to decrease over time of storage, reaching 50% after 72 hr. Decreased sperm motility seemed to follow the same trend as sperm viability. Sperm traits resulted to be very similar both in size and in shape with those of “Lessonae” frog group, one of the lineages of Western Palearctic species complex, suggesting a strong phylogenetic relationship among these species.     

Taxonomic importance of pollen morphology in Veratrum L. (Melanthiaceae) using microscopic techniques

Previous studies reported that intrageneric relationships of genus Veratrum of family Melanthiaceae are controversial and hard to delimit. Therefore, we observed the pollen morphological features of six species in the genus Veratrum in detail using both light microscopy and scanning electron microscopy and investigated their significance for Veratrum taxonomy. Among them, five were studied for the first time. The results demonstrated that pollen grains of Veratrum are medium in size with P/E being 0.31–0.60. Three types of shape in polar view have been observed elliptic, long‐elliptic, or wide‐elliptic with blunt or rounded at both ends. Two types of width of colpus reported narrow or wide, two types of depth of colpus reported deep or flat, and two types of length of colpus reported extend almost or do not extend to the ends, whereas two types of colpus membranes reported absent or obvious. One type of surface ornamentation was noted as reticulate. These results support species Veratrum album and Veratrum lobelianum as well as Veratrum grandiflorum and Veratrum oxysepalum as two independent species, respectively, rather than classifying Veratrum into two sections. Overall, we demonstrated that the ratio of polar axis length to equatorial axial length, pollen characteristics at the polar view, the colpus morphology, and the surface ornamentation of pollen grains of genus Veratrum have important systematic significance in identification and delimitation of species.     

Using microscopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranunculaceae)

In present research, micromorphological characters of the genus Delphinium L. (sensu lato) Ranunculaceae seeds and fruits were studied using microscopic techniques. A total of 37 species were studied using light (LM) and scanning electron microscopy (SEM) to evaluate seeds and fruits micromorphological characteristics for the taxonomic identification and species relationship in the genus. For each taxon, 33 characters were observed and studied. All characters were analyzed using MVSP software and UPGMA method by using dissimilarity index (average taxonomic distance). Considerable differences were observed in trichomes shape of fruit and seed color features. The results revealed that all studied taxa are separated into three clades. The first clade consists of D. pallidiflorum and D. semibarbatum, which have black seed and two types of glandular and crispate trichomes in fruit. The second clade consists of D. speciosum, D. szowitsianum, D. dasystachyson, D. aquilegifolium, D. ursinum, C. trigonelloides, and C. camptocarpa, which had a brown color seeds, two types of glandular and villous trichomes on fruits also have been observed. The remaining species were classified as third clade having various colors including yellow, black, and brown color seeds, the trichome shapes included one of the following forms strigose, villous, trichomes less, glandular, hooked, and crispate trichomes in fruits. The aim of the present study is to use micromorphological characters of seeds and fruits for the taxonomic identification and species delimitation in the genus Delphinium.     

Ultrastructure of larvae and puparia of the blowfly Chrysomya megacephala (Diptera: Calliphoridae)

Chrysomya megacephala is a forensic important fly, and its immature forms also cause myiasis. The adults are the first insects to reach a carcass and can oviposit just a few hours after arrival. Therefore, the knowledge of immature stages of flies is essential for correct identification of the species found on corpses. Scanning electron microscopy (SEM) gives detailed information on the morphological characters that can help identify the immature forms of flies. The aim of this study is to identify larvae and puparia of C. megacephala using SEM. The larval instar body of C. megacephala is similar at all instars. The integument is smooth with small spines located at the limit of all segments. The cephalic region has a group of robust spines with one or two tips. The puparia are very similar to third instar larvae, except for the cephalic structures that are retracted. The integument shows the wrinkles from the third instar larvae and posterior spiracle disc with three spiracular openings localized on the top of an elevation. In conclusion, SEM provides some characteristics to distinguish among Chrysomya species that could help entomologists to identify immature found on corpses.