Effects of Preservative Concentrations on Larval Cephalopharyngeal Skeleton of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae), an Alternative Indicator to Larval Body Length for mPMI Estimation

Introduction: In forensic entomology, dipterous larval specimens found feeding on decomposing corpses are usually preserved in a range of 70-95% ethanol before being subjected to minimum postmortem interval (mPMI) assessment. However, larval body size, which is commonly used to infer mPMI, can be affected by preservatives resulting miscalculation of estimation. This study compared the effect of ethanol concentrations on larval body and cephalopharyngeal skeleton, a potential substitute to estimate larval age for mPMI estimation. Methods: Experiments were conducted on homogenised colony of Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) third instar larvae reared in similar conditions. They were fixed with hot water (»80ºC) and preserved in 70% and 90% ethanol. After seven days in preservatives, larvae were measured for total body length from furthest part of the head to the last abdominal segment. Cephalopharyngeal skeleton was subsequently extracted from the body and measured based on morphometric landmarks on the pharyngeal sclerite. Centroid size of the cephalopharyngeal skeleton was also calculated based on the configuration of five morphometric landmarks. Results: In all four study replicates, pairwise comparisons with the original size indicated that larval body length was significantly affected by ethanol concentration (p<0.001) whilst only two of the study replicates showed cephalopharyngeal skeleton maintained its size when preserved in different ethanol concentration. Conclusion: Possible causes of variations are discussed herein with the results clearly indicated cephalopharyngeal skeleton should be considered as alternative growth parameter for mPMI estimation.

Taxonomic notes on the genus Campiglossa Rondani (Diptera, Tephritidae, Tephritinae, Tephritini) in India, with description of three new species

Three new species of Campiglossa Rondani are described from India: adults of both sexes and third instar larvae of C. ialong David, Salini &amp; Hancock, sp. nov. and C. sherlyae David &amp; Hancock, sp. nov., plus an adult female of C. shaktii David, Sachin &amp; Hancock, sp. nov., are described and illustrated. Postabdominal structures, cephalopharyngeal skeleton, and anterior and posterior spiracles of C. gemma (Hering, 1939) and C. sororcula (Wiedemann, 1830) are illustrated. DNA barcode sequences of C. ialongsp. nov., C. sherlyaesp. nov., and C. gemma were obtained and reported. Records of C. absinthii (Fabricius, 1805) and C. iracunda (Hering, 1938) are regarded as misidentifications of C. lyncea (Bezzi, 1913) and C. shaktiisp. nov., respectively, and excluded from the Indian fauna. A key to the known species of Campiglossa from India is provided. Results of preliminary phylogenetic analysis using COI revealed that C. ialongsp. nov. is paraphyletic to the Campiglossa misella group and C. C. sherlyaesp. nov. is a sister species of C. deserta.

Chrysomya megacephala (Fabricius, 1794) (Diptera: Calliphoridae) development by landmark-based geometric morphometrics of cephalopharyngeal skeleton: a preliminary assessment for forensic entomology application

Background Considering the practicality of geometric morphometrics which could discriminate insect species, this application was extended to the analysis of blow fly larval growth based on cephalopharyngeal skeleton. In forensic entomology, cephalopharyngeal skeleton plays a crucial role in species identification but the morphometric information of this part is scarce. In this study, Chrysomya megacephala (Fabricius, 1794) was reared in two study replicates in natural conditions and samplings were conducted at fixed daily intervals. Cephalopharyngeal skeletons were removed from larvae and mounted on glass slides. Images were obtained from the specimens; digitized and geometric morphometric analysis on C. megacephala cephalopharyngeal skeletons was performed with MorphoJ software based on the ordination of five landmarks. The assessments of this analysis were based on centroid size measurements, visualization on the landmarks displacements, classification of the relative landmarks by using canonical variate analysis, and ontogenetic allometry determination. Findings Centroid size was strongly correlated with developmental time (p < 0.05) and significantly different between daily intervals (p < 0.05). Ontogenetic allometric effect based on multivariate regression on Procrustes coordinates and centroid size was significant (p < 0.0001), indicating that shape was influenced by growth (60.3%). Disposition occurred on all landmarks during development and was further discriminated based on age groups. Conclusions Other than discriminating between species, geometric morphometrics was found to be practical to visualize larval growth based on cephalopharyngeal skeletons which can be useful in forensic entomology.

Ultramorphological Characteristics and Development Time of Immature Stages of Piophila casei (Diptera: Piophilidae)

Piophila casei (Linneus), known as the cheese skipper fly, is a sarcosaprophagous dipteran, meaning it has a wide range of feeding habits: proteins, decaying animal and vegetable matter, and feces. The biological aspects of P. casei are relevant to several fields, including forensic entomology and human and veterinary medicine. This work presents the ultrastructure characteristics of P. casei, compares the structures present during its development using light microscopy and Scanning Electron Microscopy (SEM) and reports the development time of eggs, larvae and pupae under controlled laboratory conditions (23°C, 60% relative humidity and 12 h of light). Colony of adults previously maintained in the laboratory (third generation) was used in this study. The morphology of the insect and of structures such as the cephalopharyngeal skeleton, the anterior and posterior spiracles and the structures and organs of the head, thorax and abdomen of the first, second and third instar larvae were characterized. The total development time of the egg from laying until the emergence of the adult was 448 hours (18.66 days); this included 8 hours for embrionary development, 248 hours for larval development and 192 hours for pupal development.Ultraestrutura e tempo de desenvolvimento de estágios imaturos de Piophila casei (Linneus)Resumo. Piophila casei (Linneus), conhecida como “cheese skipper fly”, é uma mosca sarcosaprófaga, possui habito alimentar diverso. Pode alimentar-se de proteínas diversas, matéria animal e vegetal em decomposição e fezes. Os aspectos biológicos de P. casei são relevantes principalmente para a entomologia forense, medicina humana e veterinária. Este trabalho apresenta características da ultraestrutura de P. casei, comparando as estruturas dos três instares larvais do desenvolvimento, utilizando microscopias de luz e eletrônica de varredura (MEV), além de descrever o tempo de desenvolvimento de ovos, larvas e pupa sob condições controladas de laboratório (23 °C, 60% humidade relativa e 12 h de luz). A morfologia geral do inseto e estruturas como o esqueleto cefalofaríngeo, os espiráculos anterior e posterior, as estruturas e órgãos da cabeça, tórax e abdômen da larva de primeiro, segundo e terceiro instares foram caracterizadas. O tempo total de desenvolvimento de ovo até a emergência do adulto foi de 448 horas (18,66 dias), sendo 8 horas para desenvolvimento embrionário, 248 horas para desenvolvimento larval e 192 horas para desenvolvimento pupal.

Morphology of preimaginal stages of Minettia longipennis Fabricius (Diptera: Lauxaniidae) MAREK SEMELBAUER & MILAN KOZÁNEK (Slovakia)

Scanning electron microscopy documentation of all preimaginal stages of the lauxaniid species Minettia longipennis (Fabricius) is presented for the first time. Images of the following morphological structures of all larval instars are provided: pseudocephalon, antenna, maxillary palpus, facial mask, cirri, mouth hooks, ambulatory lobes, anterior and posterior spiracles. Illustrations of the cephalopharyngeal skeleton for all three instars in lateral, ventral and dorsal views are provided. All instars are clearly separable by morphological features. The first instar has unique modification of the facial mask and structure of the cephalopharyngeal skeleton. Second and third instars are similar in overall form and differ in details in structure of the mouthooks and anterior spiracles, and by the number of pharyngeal ridges.

Embryonic head involution and rotation of male terminalia require the Drosophila locus head involution defective.

We have characterized the head involution defective (hid) locus which is located within the chromosomal region 75B8-C1,2. During the morphogenetic reorganization of the embryonic head region, hid+ function is necessary for the movement of the dorsal fold across the procephalon and clypeolabrum, a process that forms the frontal sac. The absence of the frontal sac in the hid mutant embryos affects the formation of the dorsal bridge and disrupts the development of the larval cephalopharyngeal skeleton. In addition to its embryonic role, this same hid function is also required during pupal development for the 360 degrees rotation of the male terminalia about the anterior-posterior body axis, and for a late step of wing blade morphogenesis. Although the abnormal wing phenotype caused by the Wrinkled (W) mutation is quite different from the one resulting from the loss-of-function hid mutations, the characterization of EMS-induced W revertants reveals that W is actually an antimorphic allele of hid.

Biology and Immature Stages of Tetanura pallidiventris, a Parasitoid of Terrestrial Snails (Dipt. Sciomyzidae)

Tetanura pallidiventris (Fallén) (Dipt. Scionyzidae) lives in terrestrial habitats in northern and central Europe, where the larvae feed on snails of the genera Cochlicopa, Discus, and Retinella. Field and laboratory data on the ecology and behaviour of the species are presented. The egg, third-instar cephalopharyngeal skeleton, and puparium are described and figured. The species is distinguished from T. fallenii Hendel, and the systematic position of the genus Tetanura Fallén is discussed.