3.5 Gene families associated with adaptive evolution
Some key gene families, including the Globin related to oxygen transport, the JHAMT and caspase 7 related to regulating development and metamorphosis, the GSTs and CYP450s related to detoxification were identified to illuminate the adaptation of Chironomid larva to benthic environment and metamorphosis mechanism.
Globin for benthic adaptation.It is known to all, most invertebrates have copper containing Hemocyanin and non heme iron Hemoglobin. Except for a few annelids, the Chironomid midge is one of the few invertebrate with Hemoglobin (Strand et al., 2004). Hemoglobin can provide necessary dissolved oxygen for metabolic activities of organisms (Bergtrom et al., 1976). Most chironomid larvae live in aquatic substrate with low dissolved oxygen and are urgent for hemoglobin to meet oxygen deficiency. Globins are the key dissoxygen carriers after binding to heme. The evolution of Globin in chironomid midges can be inferred by sequencing the genome of C. striatipennis . We identified were 10 Globin genes of C. striatipennis . The 10 Globin can be divided into three subfamilies based on ncbi database, seven in the subfamily Globin Ⅲ, two in the subfamily Globin Ⅶ and the other one in the subfamily Globin Ⅹ (Figure 5-a). It should be noted that eight Globin genes belonging to three gene tandem clusters in the genome have different gene sequences and number of conserved functional regions, which indicates that the gene has experienced some convergent or divergent mutation after tandem expansion.
Considering in early larval stages, C. striatipennis shifts from pelagic to benthic habitat in water and then emerges on water surface, in order to verify the hemoglobin produced by porphyrin pathway to adapt to the situation of low dissolved oxygen in benthic habitat at larval stage, 8 genes or gene family members involved in porphyrin metabolic pathway were analyzed in whole life cycle. These genes include 5-aminolevulinate synthase (ALAS), porphobilinogen synthase (ALAD), hydroxymethylbilane synthase (HMBS), uroporphyrinogen Ⅲ decarboxylase (UROS), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen Ⅲ oxidase (CPOX), protoporphyrinogen Ⅲ oxidase (PPOX) and protoporphyrin ferrochelatase (FECH) (Frankenberg et al., 2001; Saffarini et al., 1991). These genes expressed significantly in larval benthic stage and began to close in pupal stage (Figure 7, Supplementary Table15,17). In addition, in C. striatipennis , among the 10 putative Globin gene family members, 8 genes which average fragments per kilobase of transcript per million mapped (FKPM) reads for Globin genes were 6.74-35492.96 were highly expressed in larval stage, but the expression level of most Globin genes decreased to less than 1% in pupal stage and 2 pseudogenes were not expressed in the whole life cycle (Figure 5-b, Supplementary Table 16).
These data provided convincing demonstration for the correlation between the differential expression of hemoglobin and the adaptation of chironomid larva to benthic life, which indicates the chironomid species with the same lifestyle as C. striatipennis have a highly effective oxygen transport system to adapt to the low dissolved oxygen in benthic habitat.
Detoxification. The insect can secrete a variety of enzymes by detoxification system to resist the influence of external poisons (Li et al., 2018). Generally, the detoxification system usually goes through three phases to resist external poisons (Epel et al., 2008; Martinez-Paz, 2018; Smital et al., 2004). However, no matter which phase is involved, Cytochrome P450s (CYP450s) and glutathione S-transferases (GSTs) enzymes play core roles in process of resisting the influence of external poisons. 37 CYP450s and 26 GSTs were identified in C. straitipennis in this study (Supplementary Figure 8). CYP450s not only play an importance role in detoxification metabolism and synthesis of hormones but also have an obvious phenomenon of gene family expansion. The phylogenetic analysis of the CYP450s in C. striatipennis showed they were divided into four branches: the CYP2, the CYP3, the CYP4 and the mitochondrial (Mito) (Figure 6-b). It is worth noting that most genes of the CYP450s inC. striatipennis are clustered under the branches of the CYP3 (15/37 genes) and the CYP4 (13/37 genes). The genes involved in these two gene subfamilies are often induced to express by exogenous toxic substances (David et al., 2010; Ffrench-Constant, 2013). More interestingly, P. vanderplanki is known as a “trisomic” creature in biological world that can survive in various extreme environments, and the number of CYP450s genes has been amplified to as high as 154 (Figure 6-a). Aboving results provide solid evidence that in chironomid midge CYP450s, especially the CYP3 and the CYP4 gene subfamily, are related to adapt to harsh benthic environment, resist interference of various toxic substances and survive successfully in some extreme environment (Lu et al., 2021).
Development. Chironomid midge has relative developmental plasticity. It can change the developmental trajectory to deal with different environments, which is also a strategy for adapting the environment. Like most insects, chironomid midge is also coordinately regulated by ecdysone and juvenile hormone from larva to pupa and to adult, juvenile hormone can prevent metamorphosis induced by ecdysone (Guo et al., 2019; Hu X. L. et al., 2019; Miki et al., 2020; Santos et al., 2019). The genes involved in biosynthesis of ecdysone and juvenile hormone which regulate insect development were also recognized inC. striatipennis. Notably, the key genes, JHAMT, involved in biosynthesis of juvenile hormone are substantially expanded. C. striatipennis harbors 43 JHAMT genes; the number is similar to P. vanderplanki (38 JHAMT) and much higher than the equivalent in other species in Chironomidae (Supplementary Figure 10).
It has been reported that there were very few chironomid midges can grow rapidly in the cold winter (Armitage et al., 1995). Most chironomid midges overwinter at the fourth larval instar and will not emerge into adults until the beginning of the next spring (Armitage et al., 1995). By foregoing reason, it is speculated that the expansion of JHAMT genes is a general strategy for chironomid to regulate the content of juvenile hormone to overwinter. Therefore, when the external situation is appropriate, the expression of JHAMT genes will decrease.
The transcriptomes were sequenced to reveal how chironomid regulates the synthesis of ecdysone and juvenile hormone during the process of molting, metamorphosis and growth in C. striatipennis . In present study, the expression level of various enzymes related to the biosynthesis of juvenile hormone decreased during the shift from larva to adult in C. striatipennis ; it is consistent with the hypothesis that the juvenile hormone of chironomid midge decreases before eclosion to ensure a smooth eclosion (Figure 7). Interestingly, the expression level of JHAMT enzyme closely related to the synthesis of juvenile hormone Ⅲ slightly increased during the shift from pupa to adult, which may be due to chironomid midge needs to provide the time required for morphology and organelle remodeling during metamorphosis (Figure 8, Supplementary Table 18).
In addition, the expression level of ecdysone 20-monooxygenase involved in the regulation of ecdysone decreased during the period of eclosion. In conclusion, C. striatipennis controls the emergence time by flexibly adjusting the concentration of juvenile hormone, which displays development plasticity in chironomid midge.
Metamorphosis mechanism. Programmed cell death plays an important role in removing cells that are unnecessary or potentially dangerous to organisms, and apoptosis is one of the most important ways of programmed cell death (Baehrecke, 2002; Fuchs & Steller, 2015; Galluzzi et al., 2018; Hay et al., 2004). Apoptosis is essential not only to maintain homeostasis and immune response but also to its metamorphosis. Apoptosis can reshape the whole morphology of organisms for metamorphosis development to help them adapt to the new environment in the future. Previous studies have shown that ecdysone can regulate the metamorphosis of D. melanogaster by inducing the expression of apoptotic effectors. The specific molecular mechanism is that ecdysone activates the expression activity of inducing downstream apoptosis factor by binding to ECR/USP heterodimer, which initiates the caspase pathway of apoptosis by inhibiting the expression activity of apoptosis inhibiting factors (Hay & Guo, 2006; Hay et al., 2004). In present study, some key components involved in these pathways were found in C. striatipennis . Caspase 7, the effector of apoptosis, was significantly expanded in the genome of C. striatipennis(Supplementary Figure 9). Furthermore, the JNK-dependent apoptosis pathway is more active in C. striatipennis , which is not likeD. melanogaster heavily relying on ecdysone to open the caspase pathway.
JNK pathway is an important signaling pathway involved in many life processes such as cell morphology construction, cell polarity establishment and apoptosis in cells (Kuranaga et al., 2002). In Diptera, the main role of JNK pathway is to promote thorax closure by assisting actin to pull bilateral thorax to the midline through apoptosis unnecessary tissue cells (Marti´n-Blanco et al., 2000; Zeitlinger & Bohmann, 1999). In this study, the expression level of components related to regulating JNK pathway ascended gradually with the advancement of metamorphosis in C. striatipennis (Figure 7, Supplementary Table 19). This means that wing shaping is the most important event in the process of C. striatipennis metamorphosis. In addition, similar as other arthropods, C. striatipennisconstructs the first line of defense through the chitin shell, which can help chironomid avoid virus or various mechanical damages. The thorax closure can promote a pair of wings to move towards the midline and close to each other. Limited by the size of the individual, the original chitin pupal cortex was inevitably partially degraded in the process of thorax closure. Therefore, the gene encoding chitinase in the genome ofC. striatipennis was also significantly expanded (Supplementary Figure 11). Interestingly, these chitinases have relative cascade effect in expression process, which was, some chitinases were extremely expressed during the transition from larva to pupa, while others were actively expressed during the shift from pupa to adult (Supplementary Table 20). These proteins work together in series with several signal pathways to promote the successful completion metamorphosis and development in C. striatipennis .