2.1 Study area
Our study was conducted in Xinjiang Uygur Autonomous Region, center of
Asia-Europe continent (Fig. 1). This region extends from the south slope
of the Altai Mountains to the hinderland of the Tianshan Mountains. The
Junggar basin between the two mountains is covered by the Gurbantunggut
desert, the second largest desert in China. This region belongs to
semi-arid and arid climatic zone, with the elevation ranging from 189 to
7435 m, and encompasses complex landforms and microclimate between
mountains and basins. Due to the characteristics of geography,
environment and climate, Xinjiang region provides biological community
with a variety of habitats and has been listed as one of the priority
areas for biodiversity conservation in China. The Irtysh River, Emin
River, Ili River and Bortala River originate from Xinjiang Uygur
Autonomous Region, northwest China (Fig. 1) and ultimately flow towards
the Republic of Kazakhstan. In particular, the Ili River is the largest
river in Xinjiang region. The Irtysh River is the only Chinese river
that disgorges into the Arctic Ocean. Ulungur Lake covers an area of
1035 square kilometers. These rivers and their affiliated water bodies
are the most important water resources in northwest China and play an
important role in maintaining the biodiversity of aquatic ecosystems
(Wang et al., 2010).
2.2 Sampling and
experimental material
Aquatic insects are absolutely predominant faunas in the transboundary
rivers of northwest China (Wang et al., 2014). Despite extraordinarily
abundant resources for aquatic insects, few studies have been carried
out in these transboundary rivers (Wang et al., 2014). Furthermore,
transboundary rivers such as the Ili River and Irtysh River
are
suffering from the intervening of anthropogenic activities, and hence
accurate classification of macroinvertebrates is essential for the
environmental and biodiversity assessment. As for the organisms used for
water quality assessment, macroinvertebrates are one of the most optimal
categories due to a large size,
weak dispersal and the sensitiveness to the change of water
quality (Resh et al., 1995).
We collected macroinvertebrate samples in four transboundary rivers (the
Irtysh River, Emin River, Ili River and Bortala River) between China and
Kazakhstan, Mongolia and Russia (Fig. 1). Study sites were set in the
main stems, tributaries and affiliated water bodies (lakes and
reservoirs).
Due to a long frozen season (November to next April), samples of Mayfly
(order Ephemeroptera), stonefly (order Plecoptera), caddisfly (order
Trichoptera), robber fly (order
Diptera), true bugs (order
Hemiptera), Coleoptera, Odonata, Mollusca and
Annelida were collected in May,
July, August and October of 2013-2017.
Macroinvertebrates were collected by
Surber net, Kick-net, Peterson grab
and D-framed dip net according to the habitat type of study areas.
Standardized sampling protocols were executed at different habitats
(i.e., lentic and lotic water bodies) from upstream to downstream.
Approximately 30,000 specimens were preserved in 95% ethanol to allow
for the morphological identification and molecular analyses. In
particular, when the color of ethanol solution for macroinvertebrate
soaking became dark yellow during the field survey, ethanol
needed to be replaced for two or
three times in order to warrant a good preservation of the specimens for
further molecular analysis. Macroinvertebrate samples were sorted and
identified under a dissecting
microscope in the laboratory, and stored at -20 °C at the College of
Fisheries, Huazhong Agricultural
University (China). In the lab, specimens were identified based on
morphological characteristics, and then intact individuals were selected
to conduct the DNA barcoding analysis for nine taxonomic groups
(Ephemeroptera, Plecoptera, Trichoptera, Diptera, Hemiptera, Coleoptera,
Odonata, Mollusca and Annelida).
2.3 DNA extractions,
amplification and sequencing
Total genomic DNA was extracted from legs, cerci, half or whole body
according to a specimen’s size,
using the phenol–chloroform protocol (Barnett and Larson, 2012). and
quantified in a NanoDrop 2000c (Thermo Scientific, USA). The primer pair
LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198
(5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al., 1994) was used to
amplify the fragment DNA of COI (mitochondrial cytochrome C oxidase I)
with the length of about 658 bp. Polymerase Chain Reactions (PCR) were
performed in 50 µL volumes using the following reagents and
concentrations: a volume of 50
μL
containing 10×PCR buffer 5 μL, Mg2+ (2.5 mmol/L) 5 μL,
dNTP (2.5 mmol/L) 3.5
μL,
l.5 μL (10 nmol/L) each primer, 0.5 U Taq polymerase (Takara, Dalian,
China),
2
μL (50 ng/μL) DNA template and complete ddH2O to 50 μL.
The PCRs were run as follows: 94 °C for 3 min; then 94 °C for 1 min, 45
°C for 2 min, and 72 °C for 3 min, for 40 cycles; and 72 °C for another
5 min (Folmer et al. 1994). PCR
products were checked by electrophoresis at 1% agarose with an
ethidium bromide stain, and if
successful, the PCR products were subsequently purified and directly
sequenced by Invitrogen Corporation in China.