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 .