Protein databases at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov) were searched using ‘major intrinsic protein,’ ‘insect,’ and ‘aquaporin’ as text queries. A total of 251 amino acid sequences were. Of these, 206 sequences were excluded from further analyses because they were short partial sequences or corresponded to putative proteins, or were redundant sequences that contained point mutations with respect to a sequence already included in the analyses. The 45 AQP proteins included in the phylogenetic analyses are listed in Table I.

Redundant sequence data were excluded using a sequence identity cut-off of 100% (http://weizhong-lab.ucsd.edu/cdhit_suite/cgi-bin/). Sequence data were aligned using the default options of ClustalX (18) and the result of multiple alignment was refined by eye. From alignment, the gap results were treated as missing data. Through the phylogenetic analyses, ambiguous alignments with highly variable regions were excluded. NJ (Neighbor-joining) analysis (19) of the amino acid alignment was based on the current distance matrix calculate the matrix Q. Robustness of the NJ tree was tested by bootstrap analyses (as implemented in MEGA7.0) (20) with 1,000 pseudo-replications.

Four major groups produced by the NJ analysis were studied further. Every group was analyzed separately. The amino acid sequences of each group were aligned using ClustalX. A consensus sequence for each group was inferred using JalView (21). Robustness of the phylogenetic results was tested by bootstrap analyses with 1,000 pseudoreplications (as implemented in MEGA7.0) and the quartet puzzling method (with 1,000 puzzling steps).

Estimated the conserved regions of each group for the water channel protein were inferred using the rates variation option in MEGA7.0, which includes Maximum Likelihood analysis and NJ tree.

Some insects are able to live in extreme environments with little to no water or periods of drought conditions. On the other hand, some of the insects that feed on blood or plant juices are able to take on too much fluid, which is associated with a particular functional water channel protein (19). An NJ analysis of 446 positions within the AQP protein were analyzed to produce an unrooted phylogenetic tree showing that insect members of the AQP family can be classified into four major groups (Fig. 1).

The first group of insect AQP includes aquaglyceroporin, Aea AQP and AQPcic, which have homology with the AQPs found in Belgica antarctica, T. septentrionalis, A. echinatior, and Culex quinquefasciatus. This group is referred as Rpips for Belgica antarctica protein. The Rpip group was found to be functionally characterized in the filter chamber of Cicadella viridis (22). At least four AQPs in the Rpip group are obvious in the genome of Aedes aegypti, the yellow fever mosquito, but only one has been functionally characterized. The result of AQP expressed in Xenopus oocytes showed that AQPcic had a higher water permeability than human AQP1 and was reversibly inhibited by Hg²⁺. Furthermore, the water permeability of Aea AQP is even greater degree than AQPcic and is reversibly inhibited by HgCl₂ when expressed in Xenopus oocytes (23).

The second group of insect AQPs include the characterized D. melanogaster protein known as Drosophila integral protein, or Drip, and is therefore referred to as the Drip group (24). D. melanogaster Drip (Dm Drip) is most similar to the mammalian AQP4 isoform, which has been functionally expressed in Xenopus oocytes and acts as a water-specific AQP (25). As well as having representatives from each insect genome, this family of AQPs also contains AeaAQP from the Dufourea novaeangliae and AngAQP from Atta colombica. Currently, the Drip group appears to be restrained to insects, although it is conceivable that these highly unusual Drip-like AQPs may be identified in other invertebrates in the future. The expression of Drip mRNA in Xenopus oocytes and yeast secretory vesicles shows that the DRIP protein channel has a very high permeability to water (26), implicating its role in the regulation of water permeability. So far, only a small part of the water channel protein function has been verified.

The third group of insect AQPs includes Aea AQP and Ang AQP and contains a representative from the insect genome research. Most of the AQP proteins in this group have a high degree of water-selectivity. This group was referred to as the Prips for P. rufa integral protein (27). No other homologous proteins have yet been characterized. This is the least characterized family of the insect AQPs and deserves further investigation (23).

The fourth group of insect AQPs contains the D. melanogaster AQP Big Brain (Dm Bib) (28), as well as Bib-like homologues from each of the other insect species for which complete genome data are available. As the function of the Dm Bib genome has been characterized, this insect AQP-like family was referred to as Bibs appropriately. When expressed in Xenopus oocytes, Dm Bib is the only characterized Bib to be found to date and functions as a non-selective cation channel; its permeability is apparently regulated by tyrosine-kinase activity (29). In addition, compared to all other known AQPs, Dm Bib has an extended C-terminal tail with a high number of putative phosphorylation sites. An all Bib homologues alignment reveals four stretches of the tail with high conservation across all insect species examined. Within these regions are five fully conserved and two semi-conserved tyrosine residues. Dm Bib plays an important role in determining Drosophila neural fate. Bibs sequences found across other insects play a similar functional role, and one of the conserved or semi-conserved tyrosine residues confers functional regulation in the tail (24).

All AQP proteins show relatively conserved motifs and overall molecular structure along the multiple alignment. The first motif contains six membrane-spanning segments with five connecting loops connecting the inside and outside of the channel (for example, accession Q9NHW7.2 of Aedes aegypti AQP is shown in Fig. 2). Two motifs are NPA (Asp-Pro-Ala) signature motifs (30). These two NPA motifs are the most commonly conserved feature among AQPs and likely play an important role in the structure and function of water channel proteins. A SG-H-NPAVT-G and TG-MNPAR-G motif that is highly conserved in the Rpips clade is replaced by SG-H-NPAV-IS and P-LNPAR-GP motifs and in the Drips clade, the SGCH-NPAV-G and SM-NPAR-GP motifs in the Prips clade, and the V-NPAV-Q and S-NPAR-P motifs in the Bibs clade. Other consensus residues among AQPs are presented in Fig. 3. Most of them are localized in the six transmembrane domains and are likely involved in the selectivity of the channel. Because the full-length sequences of insect AQPs are about 300 bp, special constraints prevent us from depicting the full multiple alignment results in the text. Instead, we chose the most important conserved region, which is the NPA area, and displayed the two conserved region sequence comparison results of insect AQP. We can also identify the homology and evolutionary relationship between them. The different specific conserved sequence has already been responsible for the functional properties (19). Despite many advances in understanding mammalian AQPs since Peter Agre's initial discoveries, not much has been done to understand the evolutionary relationships among insect AQPs. We undertook a detailed analysis of AQP amino acid sequences to construct a phylogenetic tree that could serve as the basis for ongoing evolutionary studies. The evolutionary relationships presented here among AQPs assist with the correct identification of AQP family members moving toward further functional investigation. The insect AQP phylogenetic tree will allow for the potential manipulation of insect evolution, such as introducing water resistance and drought-resistant properties. During the development of mammalian follicles, the formation and maturation of follicles, the transport of follicular fluid, the formation of antral cavities, and the atresia of follicles are closely related to the regulation and expression of AQPs.

In summary, the evolutionary relationships presented here among AQPs assist with the correct identification of AQP family members toward the goal of further functional investigation (31). The insect AQP phylogenetic tree presented here will allow for the potential manipulation of insect evolution, such as introducing water resistance and drought-resistant properties (32). In addition, this study provides a basis for further research on the structure and function of insect sequences (33).