Freshwater species of the Seychelles

Study Extent

The Republic of Seychelles

Sampling

15 principal perennial rivers are selected based on their importance in terms of length, size of water catchment and protected status, and their accessibility. These rivers will be surveyed once a year (minimum) twice a year during (North-west Monsoon and South-east Monsoon) using electrofishing methods, SASS (South African scoring system) netting, eDNA sampling and analysis where electrofishing may not be possible.

Method steps

1. Sampling design for data collection 1. Electrofishing method and barcoding for freshwater fish survey Electrofishing Electrofishing will be led by Gaea Seychelles and experts from MNHN. A generator (Dekka Lord 2000) will be used for the sampling of freshwater fishes. The portable machine that was used had a battery with an output power of 180 W. It gives rectangular impulses at a fixed frequency of 100 Hz or 400 Hz. The duty cycle is controllable and is of 5 to 25 %. It has three voltage outputs: 150, 200 and 300 V. Electric fishing is performed in wadable streams by progressing in an upstream direction; in that way the water stays clear in front of the operators. The method (non-lethal) consists in placing a fishing electrode near habitat shelters in which the animals are found; the electrode creates an electrical field, which has an attraction effect within a radius of a one-meter zone under average conditions. When a fish comes within this field, it is stunned; it can then be caught easily with a hand net. Snorkeling with mask and hand nets will be also used, if possible, as a complementary method to the electrofishing. Each species caught is identified on the field and released. To constitute DNA barcoding reference libraries a piece of fin will be taken while the fish will be anaesthetised (the fish is awakened in clear water before it is released). Fin clips will be stored and preserved in 95% ethanol for molecular analysis (partial COI gene). DNA analysis for freshwater fish Barcoding for fish will be done by MNHN. The adoption of Next-Generation Sequencing (NGS) by the field of DNA barcoding of Metazoa has been hindered by the fit between the classical COI barcode and the Sanger-based sequencing method. The method used is described in the paper Hinsinger et al. 2015: a framework for the sequencing and multiplexing of mitogenomes on NGS platforms that implements (I) a universal long-range PCR-based amplification technique, (II) a two-level multiplexing approach (i.e. divergence-based and specific tag indexing), and (III) a dedicated demultiplexing and assembling script from an Ion Torrent (now we use an Illumina) sequencing platform. Hinsinger et al. 2015 provides a case study of mitogenomes obtained for two vouchered individuals of fishes and show that this workflow enables to recover over 100 mitogenomes per sequencing chip on a PGM sequencer. Molecular reference libraries: towards a global biodiversity information system for species identifications The MNHN build since several years’ molecular reference libraries for freshwater fish living in Indo-Pacific islands and particularly in Indian Ocean, establishing lists of taxonomically valid species. The use of eDNA and COI barcoding relies necessary on the development of DNA barcode reference libraries for known species in order to foster automated assignment of unknown specimens to known species. For automation purposes, both stability of the reference libraries and reproducibility of the molecular identifications should be guaranteed. That’s why MNHN build these libraries studying and sequencing (when possible) types specimens of each species. The libraries have already the sequences of the gene needed for several species distributed in Seychelles. 2. Micro and macro-invertebrate sampling lankton net method: Plankton nets of 80, 125 and 200 mesh size were used in this research. Mesh size is an aperture or eye size in net. Sample are collected through two methods that is horizontal tow and vertical tow. One need to enter in water through boat trawler to collect water sample. The handling of plankton nets from margin has possibility of damage and clogging. The surface water sample is collected through horizontal tow by tying floaters on end of the ring and heavy material at other end of the ring. Volume of water that flow through net can be calculated by formula as mentioned below. lankton net method: Plankton nets of 80, 125 and 200 mesh size were used in this research. Mesh size is an aperture or eye size in net. Sample are collected through two methods that is horizontal tow and vertical tow. One need to enter in water through boat trawler to collect water sample. The handling of plankton nets from margin has possibility of damage and clogging. The surface water sample is collected through horizontal tow by tying floaters on end of the ring and heavy material at other end of the ring. Volume of water that flow through net can be calculated by formula as mentioned below. Plankton net method: Plankton nets of 80, 125 and 200 mesh size were used in this research. Mesh size is an aperture or eye size in net. Sample are collected through two methods that is horizontal tow and vertical tow. One need to enter in water through boat trawler to collect water sample. The handling of plankton nets from margin has possibility of damage and clogging. The surface water sample is collected through horizontal tow by tying floaters on end of the ring and heavy material at other end of the ring. Volume of water that flow through net can be calculated by formula as mentioned below. Micro and macro-invertebrates sampling and identification will be done in conjuction with our expert partner GroundTruth (South Africa). Micro and macro-invertebrates will be collected using the methods described in the South African Scoring System version 5 (SASS5, Dickens and Graham 2002 ), a rapid kick sampling technique designed for wadable rivers that is ISO 17025 accredited. Kick samples are collected from three major biotopes 1) Stones (2cm – 25cm and includes bedrock sheets) in and out of current, 2) marginal and aquatic vegetation in and out of current and 3) gravel, sand and mud. Each biotope is sampled for a specific time or area as per the SASS5 method and macro-invertebrates from each biotope are identified to a family level. Each family is prescribed a sensitivity score from 1 to 15 depending on the water quality requirements for the family. Based on the sum and averages scores per taxon an aquatic ecological condition is generated for the river. MiniSASS is a citizen science version of the SASS5 method (Graham, et al. 2004 ). For miniSASS, the combined biotopes are sampled for approximately 10 mins and a combined sample is identified down to an order level. Each order has been assigned a sensitivity rating depending on the average water quality requirements of the Order and the average score per Order is used to determine the river condition. Plankton nets will be used to horizontally sweep through the water to collect zooplankton in the 15 rivers. The net will be rinsed to wash its content into the storage tube. The Zooplankton will be identified using a microscope in the lab.

1. Arthropoda
   rank: phylum
2. Bulinidae
   rank: family
3. Eleotridae
   rank: family
4. Gyrinidae
   rank: family
5. Crambidae
   rank: family
6. Kuhliidae
   rank: family
7. Decapoda
   rank: order
8. Noctuidae
   rank: family
9. Aeshnidae
   rank: family
10. Mugilidae
    rank: family
11. Culicidae
    rank: family
12. Hemiptera
    rank: order
13. Tingidae
    rank: family
14. Hydroptilidae
    rank: family
15. Anguillidae
    rank: family
16. Microdesmidae
    rank: family
17. Sesarmidae
    rank: family
18. Gymnophiona
    rank: order
19. Osphronemidae
    rank: family
20. Sooglossidae
    rank: family
21. NOT ASSIGNED
    rank: family
22. Chordata
    rank: phylum
23. phylum
    rank: phylum
24. Pomatiidae
    rank: family
25. Pomatiopsidae
    rank: family
26. Ceratopogonidae
    rank: family
27. Nolidae
    rank: family
28. Canacidae
    rank: family
29. Ambassidae
    rank: family
30. Limoniidae
    rank: family
31. Hydrophilidae
    rank: family
32. Moringuidae
    rank: family
33. Varunidae
    rank: family
34. Ecnomidae
    rank: family
35. Poeciliidae
    rank: family
36. Mugiliformes
    rank: order
37. Ephemeroptera
    rank: order
38. Erebidae
    rank: family
39. Helicopsychidae
    rank: family
40. order
    rank: order
41. Nabidae
    rank: family
42. Aplocheilidae
    rank: family
43. Neritiliidae
    rank: family
44. Amphibia
    rank: class
45. Gastropoda
    rank: class
46. Lepidoptera
    rank: order
47. Indotyphlidae
    rank: family
48. Coenagrionidae
    rank: family
49. Cyprinidae
    rank: family
50. Hydrometridae
    rank: family
51. Cichlidae
    rank: family
52. Mollusca
    rank: phylum
53. Succineidae
    rank: family
54. Diptera
    rank: order
55. Elmidae
    rank: family
56. Pulmonata
    rank: order
57. kingdom
    rank: kingdom
58. Ephydridae
    rank: family
59. Muscidae
    rank: family
60. Trichoptera
    rank: order
61. Cycloneritida
    rank: order
62. Animalia
    rank: kingdom
63. Perciformes
    rank: order
64. Limnichidae
    rank: family
65. Anura
    rank: order
66. Stratiomyidae
    rank: family
67. class
    rank: class
68. Veliidae
    rank: family
69. Leptophlebiidae
    rank: family
70. Corduliidae
    rank: family
71. Gobiidae
    rank: family
72. Littorinimorpha
    rank: order
73. Libellulidae
    rank: family
74. family
    rank: family
75. Cyprinodontiformes
    rank: order
76. Neritidae
    rank: family
77. Coleoptera
    rank: order
78. Odonata
    rank: order
79. Insecta
    rank: class
80. Hyperoliidae
    rank: family
81. Leptoceridae
    rank: family
82. Dolichopodidae
    rank: family
83. Atyidae
    rank: family
84. Argiolestidae
    rank: family
85. Polycentropodidae
    rank: family
86. Phoridae
    rank: family
87. Atriplectididae
    rank: family
88. Palaemonidae
    rank: family
89. Cypriniformes
    rank: order
90. Kraemeriidae
    rank: family
91.  Osteichthyes 
    rank: class
92. Cecidomyiidae
    rank: family
93. Saldidae
    rank: family
94. Ochteridae
    rank: family
95. Lestidae
    rank: family
96. Anguilliformes
    rank: order
97. Pyralidae
    rank: family
98. Actinopterygii
    rank: class
99. Hydropsychidae
    rank: family
100. Deckeniidae
     rank: family
101. Malacostraca
     rank: class
102. Stylommatophora
     rank: order
103. Ptychadenidae
     rank: family

The Republic of Seychelles A conglomerate of 115 islands situated in the Indian Ocean near the east coast of Africa