Haemocyanin is essential for embryonic development and survival in the migratory locust

Abstract

Haemocyanins are commonly known as copper-containing oxygen carriers within the haemolymph of arthropods, and have been found in many orders of insects. However, it remains unresolved why haemocyanins persist in insects that possess elaborate tracheal systems for oxygen diffusion to cells. Here we identified haemocyanins in the migratory locust Locusta migratoria that consists of two distinct subunits, Hc1 and Hc2. Genomic sequence analysis indicated that Hc1 and Hc2 have four and three gene copies, respectively, which may have evolved via gene duplication followed by divergent evolution of introns. The two subunits exhibit abundant and embryonic-specific expression at the mRNA and protein level; their expression peaks in the mid-term embryo and is not detectable in the late nymphal and adult stages. A larger proportion of the haemocyanins is present in the yolk compared with that in the embryo. Immunostaining shows that haemocyanins in the embryo are mainly expressed in the epidermis. Knockdown of Hc1 and Hc2 results in significant embryonic developmental delay and abnormality as well as reduced egg hatchability, ie the proportion of hatched eggs. These results reveal a previously unappreciated and fundamental role for haemocyanins in embryonic development and survival in insects, probably involving the exchange of molecules (eg O2 ) between the embryo and its environment.

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

“…In this stage, the embryos of many grasshopper species such as Tibetan locusts and Aulocara elliotti cease growth and enter diapause, whereas the lowland locust embryos continue to develop (Figures 4 and 10) [4]. Note that the changes caused by gene suppressed can be detected with live embryos using OCT, a clear advantage over using standard microscope and immunohistochemistry methods [24,25] in which the embryos have to get killed first. After 14 days the lowland locust embryo develops to Stage 27 and hatches (Figures 4 and 11).…”

“…The primers used for dsRNA preparation are from references [24,25]. The primers used for dsRNA preparation are from references [24,25].…”

“…The primers used for PCR were from references [24,25]. The standard curve method was used to measure the target gene mRNA relative expression levels, which were normalized by RP49.…”

“…To demonstrate the capabilities and effectiveness of OCT for evaluating the effects of gene modification in the embryos of insects, we used the OCT system described in Figure 1 to detect the phenotypic variations after the suppression of three genes in multiple embryos with numbers described below by RNA interference (RNAi), including HC1, HC2 and hunchback, which are considered key genes in the embryonic development [24,25]. The control embryos were microinjected dsRNA of green fluorescent protein (GFP) gene that is absent in locust genome.…”

“…It is therefore important to better understand issues relating to their reproduction. In addition, locusts are readily manipulated to loss-offunction by RNA interference (RNAi) technology that suppresses the gene expression [24,25]. We find that locust eggs have a shell sufficiently transparent for the light emitted from OCT to penetrate and therefore believe that they are good candidates for OCT related embryonic development research.…”