Comparisons of Respiratory Pupal Gill Development in Black Flies (Diptera: Simuliidae) Shed Light on the Origin of Dipteran Prothoracic Dorsal Appendages.

During the transformation of immature aquatic dipteran insects to terrestrial adults, the prothoracic pupal respiratory organ enables pupae to cope with flood-drought alternating environments. Despite its obvious importance, the biology of the organ, including its development, is poorly understood. In this study, the developing gills of several Simulium Latreille (Diptera: Simuliidae) spp. were observed using serial histological sections and compared with data on those of other dipteran families published previously. The formation of some enigmatic features that made the Simulium gill unique is detailed. Through comparisons between taxa, we describe a common developmental pattern in which the prothoracic dorsal disc cells not only morph into the protruding respiratory organ, which is partially or entirely covered with a cuticle layer of plastron, but also invaginate to form a multipart internal chamber that in part gives rise to the anterior spiracle of adult flies. The gill disc resembles wing and leg discs and undergoes cell proliferation, axial outgrowth, and cuticle sheath formation. The overall appendage-like characteristics of the dipteran pupal respiratory organ suggest an ancestral form that gave rise to its current forms, which added more dimensions to the ways that arthropods evolved through appendage adaptation. Our observations provide important background from which further studies into the evolution of the respiratory organ across Diptera can be carried out.

Determining the Number of Instars in Simulium quinquestriatum (Diptera: Simuliidae) Using k-Means Clustering via the Canberra Distance.

Simulium quinquestriatum Shiraki (Diptera: Simuliidae), a human-biting fly that is distributed widely across Asia, is a vector for multiple pathogens. However, the larval development of this species is poorly understood. In this study, we determined the number of instars in this pest using three batches of field-collected larvae from Guiyang, Guizhou, China. The postgenal length, head capsule width, mandibular phragma length, and body length of 773 individuals were measured, and k-means clustering was used for instar grouping. Four distance measures-Manhattan, Euclidean, Chebyshev, and Canberra-were determined. The reported instar numbers, ranging from 4 to 11, were set as initial cluster centers for k-means clustering. The Canberra distance yielded reliable instar grouping, which was consistent with the first instar, as characterized by egg bursters and prepupae with dark histoblasts. Females and males of the last cluster of larvae were identified using Feulgen-stained gonads. Morphometric differences between the two sexes were not significant. Validation was performed using the Brooks-Dyar and Crosby rules, revealing that the larval stage of S. quinquestriatum is composed of eight instars.