---
# System prepended metadata

title: Metabarcoding -- flies

---

# **Metabarcoding -- flies**


# **2023 Project**

# Workflow

**Main objective:** To collect flies from the living sites of the South American species highlighted below in yellow in order to test a more simplified technique for metabarcoding identification of Neotropical species. There are two options to place the traps: 1) close to the enclosures or 2) close to feces and other materials from the target species when they are removed. 

**Location:** Tierpark Berlin (Am Tierpark 125, 10319 Berlin)

## Sample colection 
**Target:** Flies.
**Location:** Living sites of the selected South American species.
**Time:** 40 minutes in each point.
**Collection method:** The flies will be attracted to the small butterfly cage by the use of a bait, which will be placed using a plastic container covered with mesh to prevent the flies from landing on the bait. The bait will be placed within a foldable butterfly cage with the zipper left partially open to admit flies.

* Example of a foldable butterfly cage:

![](https://hackmd.io/_uploads/SkqFik7la.png =250x250)


* Foldable butterfly cage in the field:

![](https://hackmd.io/_uploads/B18IckQx6.png =250x250)


After 40 minutes, the flies that landed inside the cage, will be captured in vials:
![](https://hackmd.io/_uploads/HkIEpyXep.png =350x250)

Obs. Each fly will be placed individually in the vials:
![](https://hackmd.io/_uploads/BJ3l8e7gT.png =350x150)

-----

Complete list of South American species in the Tierpark Berlin:
*The underlined names would be those of the species of interest.

|Class|Order|Specie|English name |State of conservation|
|---|--|----|---|--|
| **Bird**|Accipitriformes|==*Harpia harpyja*==|Harpy eagle|VU|
|||*Cathartes aura septentriona*|Eastern turkey vulture|LC|
|||*Cathartes burrovianus*|Lesser yellow-headed vulture|LC|
|||*Coragyps atratus*|Black vulture|LC|
|||==*Sarcorhamphus papa*==|King vulture|LC|
|||*Geranoaetus melanoleucus australis*|Eastern grey buzzard eagle|LC|
||Psittaciformes |==*Anodorhynchus hyacinthinus*==|Hyacinth macaw|VU|
|||*Amazona bodini*|Northern festive amazon|LC|
|||*Amazona farinosa*|Southern mealy amazon|LC|
|||==*Ara ambiguus*==|Buffon's macaw|EN|
|||*Ara ararauna*|Blue-and-yellow macaw|LC|
|||*Aratinga jandaya*|Jandaya conure|LC|
|||*Cyanoliseus patagonus*|Patagonian conure|LC|
|||*Primolius couloni*|Blue-headed macaw|VU|
||Phoenicopteriformes|*Phoenicopterus ruber*|Cuban flamingo|LC|
|||==*Phoenicopterus chilensis*==|Chilean flamingo|NT|
||Pelecaniformes|*Eudocimus ruber*|Scarlet ibis|LC|
|||*Ardea alba*|Great egret|LC|
|||*Pelecanus occidentalis carolinensis*|Eastern brown pelican|-|
|||==*Pelecanus thagus*==|Humboldt brown pelican|NT|
||Anseriformes|*Chauna torquata*|Crested screamer|LC|
|||*Spatula puna*|Puna teal|LC|
|||*Cygnus melanocoryphus*|Black-necked swan|LC|
|||*Chloephaga poliocephala*|Ashy-headed goose|LC|
|||*Chloephaga rubidiceps*|Ruddy-headed goose|LC|
|||*Neochen jubatus*|Orinoco goose|NT|
|||*Callonetta leucophrys*|Ringed teal|LC|
|||*Sarkidiornis sylvicola*|American comb duck|-|
|||*Netta peposaca*|Rosybill|LC|
|||*Amazonetta brasiliensis brasiliensis*|Brazilian teal|LC|
|||*Anas bahamensis*|White-cheeked pintail|LC|
|||*Anas georgica spinicauda*|Brown pintail|LC|
|||*Anas specularis*|Bronze-winged duck|NT|
|||*Mareca sibilatrix*|Chiloe wigeon|LC|
|||*Tachyeres patachonicus*|Flying steamer duck|LC|
|||*Tachyeres pteneres*|Magellanic flightless steamer duck|LC|
||Eurypygiformes|*Eurypyga helias*|Sunbittern|LC|
||Columbiformes|*Claravis pretiosa*|Blue ground pigeon|LC|
||Apodiformes|*Chrysolampis mosquitus*|Ruby-topaz hummingbird|LC|
||Gruiformes|*Aramides ypecaha*|Giant wood rail|LC|
|||*Psophia crepitans*|Common trumpeter|LC|
||Sphenisciformes|==*Spheniscus humboldti*==|Humboldt penguin|VU|
||Charadriiformes|*Chroicocephalus cirrocephalus poliocephalus*|Grey-hooded gull|LC|
|||*Larus modestus*|Grey gull|LC|
|||*Leucophaeus atricilla*|Laughing gull|LC|
||Strigiformes|*Bubo virginianus*|Great horned owl|LC|
|||*Strix chacoensis*|Chaco owl|NT|
|||*Strix rufipes*|Rufous-legged owl|LC|
||Piciformes|*Pteroglossus viridis*|Green aracari |LC|
||Falconiformes|*Phalcoboenus megalopterus*|Mountain caracara|LC|
|||*Falco sparverius*|American kestrel/sparrowhawk|LC|
||Passeriformes|*Euphonia violacea*|Violaceous euphonia|LC|
|||*Psarocolius decumanus*|Crested oropendola|LC|
|||*Furnarius leucopus*|Pale-legged hornero|LC|
|||*Cyanerpes cyaneus*|Red-legged honeycreeper|LC|
|||*Tersina viridis*|Swallow tanager|LC|
|||*Coryphospingus cucullatus*|Red-crested finch|LC|
|||*Ramphocelus bresilius*|Brazilian tanager|LC|
|||*Pipraeidea bonariensis*|Blue-and-yellow tanager|LC|
|||*Tangara chilensis*|Paradise tanager|-|
|||*Tangara gyrola*|Bay-headed tanager|LC|
|||*Cotinga cayana*|Spangled cotinga|LC|
|||*Xipholena punicea*|Pompadour cotinga|LC|
|||*Tyrannus melancholicus*|Tropical kingbird|LC
|**Mammalia**|Primates|*Mico argentatus*|Silver monkey|LC|
|||==*Alouatta seniculus*==|Venezuelan red howler monkey|LC|
|||*Saguinus imperator subgrisescens*|Emperor tamarin|LC|
|||*Saguinus labiatus labiatus*|Red-bellied white-lipped tamarin|LC|
|||*Saguinus midas*|Red-handed tamarin|LC|
|||==*Sapajus xanthosternos*==|Golden-bellied capuchin|CR|
|||*Pithecia pithecia pithecia*|White-faced saki|LC|
|||*Alouatta seniculus*|Red howler|LC|
||Pilosa|==*Choloepus didactylus*==|Linne's two-toed sloth|LC|
|||==*Tamandua tetradactyla nigra*==|Long-tailed tamandua|LC|
||Sirenia |*Trichechus manatus*|Caribbean manatee|VU|
||Rodentia|*Sigmodon hispidus*|Hispid cotton rat|LC|
|||==*Cavia aperea*==|Brazilian guinea pig|NT|
|||*Dolichotis patagonum*|Patagonian mara|NT|
|||*Dolichotis salinicola*|Chacoan mara|LC|
|||*Galea musteloides monasteriensis*|Muenster guinea pig|-|
|||*Dasyprocta azarae*|Azara's agouti|-|
|||*Dasyprocta leporina*|Brazilian agouti|LC|
|||*Octodon degus*|Degu|LC|
|||*Spalacopus cyanus*|Cururo|LC|
||Carnivora|==*Leopardus geoffroyi*==|Geoffroy's cat|LC|
|||==*Leopardus wiedii nicaraguae*==|Central American margay|NT|
|||==*Panthera onca*==|Jaguar|NT|
|||==*Chrysocyon brachyurus*==|Maned wolf|NT|
|||*Speothos venaticus*|Bush dog|NT|
|||==*Tremarctos ornatus*==|Spectacled bear|VU|
||Perissodactyla|==*Tapirus bairdii*==|Baird's tapir|EN|
||Artiodactyls|==*Catagonus wagneri*==|Chacoan peccary|EN|
|||*Pecari tajacu*|Collared peccary|LC|
|||==*Lama glama*==|Llama|-|
|||*Lama guanicoe*|Guanaco|LC|
|||==*Vicugna pacos*==|Alpaca|-|
|||*Vicugna vicugna*|Vicugna|LC|
|**Reptilia**|Testudines|*Chelonoidis carbonarius*|South American red-footed tortoise|-|
|||==*Chelonoidis denticulata*==|South American yellow-footed tortoise|VU|
|||*Chelus fimbriatus*|Matamata turtle|LC|
|||*Phrynops hilarii*|Spot-bellied side-necked turtle|LC|
||Squamata|*Iguana iguana*|Green iguana|LC|
|||*Epicrates maurus*|Brown rainbow boa|LC|
|||*Crotalus durissus*|Tropical rattlesnake|LC|
||Crocodilia|==*Paleosuchus palpebrosus*==|Dwarf caiman|LC|
|**Amphibia**|Anura|*Trachycephalus resinifictrix*|Mission golden-eyed tree frog|LC|
|||*Ceratophrys ornata*|Ornate horned frog|NT|


**>> Summary <<**
||Class|nº of orders|nº of species|State of conservation|
|---|---|---|---|---|
||Bird|15|64|LC (51); VU (4); EN (1); NT (5); No info (3)|
||Mammalia|7|33|LC (18); CR (1); VU (2); NT (6); EN (2); No info (4)|
||Reptilia|3|8|LC (6); VU (1); No info (1)|
||Amphibia|1|2|LC (1); NT (1)|
|**Total**|4|26|107| Lower risk (88); Threatened (11); No info (8)|
* Least Concern (LC) - Lower risk 
* Near Threatened (NT) - Lower risk 
* Vulnerable (VU) - Threatened
* Endangered (EN) - Threatened
* Critically Endangered (CR) - Threatened
----



## **Selected species**

|Class|Order|Specie|SoC|Photo|
|---|---|---|---|---|
|**Bird**|Accipitriformes|*Harpia harpyja* (Harpy eagle)|VU|![](https://hackmd.io/_uploads/ByKZH_bx6.png =160x110) Map ✅|
|||*Sarcorhamphus papa* (King vulture)|LC|![](https://hackmd.io/_uploads/rJXwrdbg6.png =150x100) Map ✅|
||Psittaciformes|*Anodorhynchus hyacinthinus* (Hyacinth macaw)|VU|![](https://hackmd.io/_uploads/rJnUNuWxp.png =150x110) Map ✅|
|||*Ara ambiguus* (Buffon’s macaw)|EN|![](https://hackmd.io/_uploads/ByrQVOblT.png =150x120) Map ✅|
||Phoenicopteriformes|*Phoenicopterus chilensis* (Chilean flamingo)|NT|![](https://hackmd.io/_uploads/BJhyE_bgp.png =150x110) Map ✅|
||Pelecaniformes|*Pelecanus thagus* (Humboldt brown pelican)|NT|![](https://hackmd.io/_uploads/Syo2X_-xp.png =150x110) Map ✅|
||Sphenisciformes|*Spheniscus humboldti* (Humboldt penguin)|VU|![](https://hackmd.io/_uploads/HyLDXdZlp.png =150x110) Map ✅|
|**Mammalia**|Primates|*Alouatta seniculus* (Venezuelan red howler monkey)|LC|![](https://hackmd.io/_uploads/H14c3v-gp.png =150x120)|
|||*Sapajus xanthosternos* (Golden-bellied capuchin)|CR|![](https://hackmd.io/_uploads/HkgRnwWe6.png =150x120)|
||Pilosa|*Choloepus didactylus* (Linne’s two-toed sloth)|LC|![](https://hackmd.io/_uploads/BJkVawZlT.png =150x120)|
|||*Tamandua tetradactyla nigra* (Long-tailed tamandua)|LC|![](https://hackmd.io/_uploads/H1nOaP-g6.png =150x120)|
||Rodentia|*Cavia aperea* (Brazilian guinea pig)|NT|![](https://hackmd.io/_uploads/SJnapwbeT.png =150x100) Map ✅|
||Carnivora|*Leopardus geoffroyi* (Geoffroy’s cat)|LC|![](https://hackmd.io/_uploads/HyQ-yOZe6.png =150x110) Map ✅|
|||*Leopardus wiedii nicaraguae* (Central American margay)|NT|![](https://hackmd.io/_uploads/ByZNkuWx6.png =150x110)|
|||*Panthera onca* (Jaguar)|NT|![](https://hackmd.io/_uploads/ryUdy_WlT.png =150x110)|
|||*Chrysocyon brachyurus* (Maned wolf)|NT|![](https://hackmd.io/_uploads/HkVpkuZl6.png =150x110) Map ✅|
|||*Tremarctos ornatus* (Spectacled bear)|VU|![](https://hackmd.io/_uploads/ByQVldZeT.png =160x110) Map ✅|
||Perissodactyla|*Tapirus bairdii* (Baird’s tapir)|EN|![](https://hackmd.io/_uploads/SyFYlubxa.png =150x110)|
||Artiodactyls|*Catagonus wagneri* (Chacoan peccary)|EN|![](https://hackmd.io/_uploads/rkYx-Obe6.png =150x110) Map ✅|
|||*Lama glama* (Llama)|-|![](https://hackmd.io/_uploads/S1gmb_WlT.png =150x110) Map ✅|
|||*Vicugna pacos* (Alpaca)|-|![](https://hackmd.io/_uploads/S1aLZOWg6.png =150x110) Map ✅|
|**Reptilia**|Testudines|*Chelonoidis denticulata* (South American yellow-footed tortoise)|VU|![](https://hackmd.io/_uploads/ByDF-_-l6.png =150x100)|
||Crocodilia|*Paleosuchus palpebrosus* (Dwarf caiman)|LC|![](https://hackmd.io/_uploads/r1t2W_-xa.png =150x100)|

**Total:** 23 species (3 classes, 13 orders), being:
* No info - 2
* Least Concern (LC) - 6 
* Near Threatened (NT) - 6 
* Vulnerable (VU) - 5
* Endangered (EN) - 3
* Critically Endangered (CR) - 1

![](https://hackmd.io/_uploads/Hy4kgafx6.png =400x450)

---
**29.09.23**
* The test with sardines didn't work, it took too long and only wasps appeared, something with a stronger smell seems to be necessary.
* Places to put the bait: various, it could be in the green area next to the animal of interest, for example, to avoid disturbing the animal. In places where the animal was in glass, we would have to check other possibilities.


![](https://hackmd.io/_uploads/HyFaGINxT.png)
*Chrysocyon brachyurus* (Maned wolf) only has a fence to separate it and a green area nearby.

![](https://hackmd.io/_uploads/H1a378Nea.png)
*Sarcorhamphus papa* (King vulture) stays with other birds, being the only one in South America.


![](https://hackmd.io/_uploads/HJ88ELVlT.png)
*Tremarctos ornatus* (Spectacled bear) was separated by glass, it would be interesting to see the possibility when cleaning, for example.

![](https://hackmd.io/_uploads/S1NGLL4x6.png)
The Harpy eagle (*Harpia harpyja*) was in a room separated by glass (as shown in the photo), but there was an "open" area next to it that appeared to be hers too.

![](https://hackmd.io/_uploads/rkOY8IEea.png)
*Lama glama* (Llama) and *Vicugna pacos* (Alpaca) are close to each other, maybe we could try a single collection point.

![](https://hackmd.io/_uploads/rk2VD8Vg6.png)
The *Anodorhynchus hyacinthinus* (Hyacinth macaw) has birds in close proximity, but it doesn't share space with other species, so placing them in close proximity could be a solution.


----
# **Sampling**
**Wednesday -- 11.10.2023**
![](https://hackmd.io/_uploads/HykmAwtbT.jpg =410x550)

![](https://hackmd.io/_uploads/r1gLRDKba.jpg =410x550)

**Thursday -- 12.10.2023**
![](https://hackmd.io/_uploads/BJ3oRwF-T.jpg)

![](https://hackmd.io/_uploads/SkTq0wK-T.jpg)

**Friday -- 13.10.2023**
![](https://hackmd.io/_uploads/SyH6AvY-T.jpg =410x550)

![](https://hackmd.io/_uploads/By4gJ_K-p.jpg =410x550)

![](https://hackmd.io/_uploads/HyCbJOYWa.jpg =410x550)

![](https://hackmd.io/_uploads/Bk6G1utbT.jpg =410x550)

![](https://hackmd.io/_uploads/S1Dmk_tbp.jpg =410x550)

---
**>> Summary <<**
|Specie| Day| Flies|
|---|---|---|
|*Speothos venaticus* (Bush dog)|11.10.23|5|
|*Chrysocyon brachyurus* (Maned wolf)|11.10.23|3
||13.10.23|4|
|*Harpia harpyja* (Harpy eagle)|12.10.23|11|
|*Anodorhynchus hyacinthinus* (Hyacinth macaw), *Ara ambiguus* (Great green macaw), *Ara glaucogularis* (Blue-throated macaw)|12.10.23|10|
|Tremarctos ornatus (Spectacled bear)|13.10.23|5|
|*Catagonus wagneri* (Chacoan peccary), *Strix cacoensis* (Chaco owl)|13.10.23|4|
|*Chauna torquata* (Sourthen screamer)|13.10.23|11|
|*Vicugna pacos* (Alpaca)|13.10.23|5|
|*Sarcorhamphus papa* (King vulture)|13.10.23|1|

**Total of flies: 40**



# :books: **Literature Research**
# 2022
:::info
:page_facing_up:	[Srivathsan et al. (2022)](https://onlinelibrary.wiley.com/doi/full/10.1111/mec.16767?casa_token=5uaMJFyh2HIAAAAA%3AMIqbvwuH20hhHvaa0KlNAIISjP2wGhY0fSKRuRE78_77Spiop_TASs4UE0mPcyU4PxUv8C2MtnXyz0k)

:movie_camera:	[Using fly poop to detect rare animals](https://www.youtube.com/watch?v=Qg7MpIVR8vA)
:::

## Sampling
* Flies were baited using fish meat of a marine fish (*Rastrelliger kanaguta*) that was obtained from supermarkets in Singapore and allowed to rot for 2–3 days in an airtight container. 
* In the field, the bait was used to attract flies using a plastic container covered with mesh to prevent flies from landing on the bait. 
* The bait was then placed within a foldable butterfly cage with the zipper left partially open to admit flies:

![](https://hackmd.io/_uploads/HkZ3F0pRh.png =250x250)

* Once flies landed on the inside of the mesh cage, they were caught using 8 ml autoclaved Sarstedt vials:

![](https://hackmd.io/_uploads/ryykjRaAh.png =200x200)

* :warning:	 To avoid contamination, new Sarstedt vials were used for each fly and gloves were worn during the collection of flies. Fly trapping was conducted for 2h at each station and between sites the butterfly cage and container were sterilized using 10% Clorox.

* Flies were transported to laboratory and frozen at −30°C --> Overall, the flies were kept alive in vials for at most 2h before being frozen. 
* Frozen flies were later removed from the Sarstedt vials by decanting and preserving them in 95% ethanol, while faecal samples and fly regurgitates on the inner walls of the tubes were classified as:

|Color | Suggesting| 
| --- | --- | 
| brown | the presence of faecal samples|
|clear | the presence of regurgitated digestive fluid|
| both| the presence of faecal samples and regurgitated digestive fluid|
| empty| -- if the tube contained nothing visible|

After noting the appearance of fly excretions, 20–50 μl* of molecular grade water was pipetted onto the fly exudes to dissolve them. 

*The amount of water was determined based amount of regurgitates or faeces observed. If vials appeared to be empty or contained amount of clear liquid similar to condensation, 20μl of molecular grade water was used to wash the inner surface of the tubes. For other samples having bigger drops of regurgitates or faeces, 50μl of molecular grade water was used.

## PCR
PCR amplifications were done using tagged primers to allow for multiplexing large number of samples on next generation sequencing platforms.

| Target| Primers| PCR conditions |
| --- |--- | ---- |
| 313-bp COI minibarcode of metazoans | m1COlintF: 5′-GGWACWGGWTGAACWGTWTAYCCYCC− 3′ (Leray et al., 2013) and modified jgHCO2198: 5′-TANACYTCNGGRTGNCCRAARAAYCA-3′ (Geller et al., 2013)|Initial denaturation at 94°C (3min) // followed by 35 cycles of denaturation at 94°C (0.5min), annealing at 45°C (1.5min), extension at 72°C (0.5min) // final extension of 72°C (3min). |
| 244-bp COI minibarcode of vertebrates| Mod_RepCOI_F: 5′-TNTTYTCMACYAACCACAAAGA-3′ and VertCOI_7216_R: 5′-CARAAGCTYATGTTRTTYATDCG-3′ (Reeves et al., 2018)|Initial denaturation at 94°C (3min) // followed by 40 cycles of denaturation at 94°C (40s), annealing at 48.5°C (2min), extension at 72°C (0.5min) // final extension of 72°C (3min)| 
| 93-bp 16S minibarcode of mammals|16Smam1: 5′-CGGTTGGGGTGACCTCGGA-3′ and 16Smam2: 5′-GCTGTTATCCCTAGGGTAACT-3′ (Taylor, 1996)|Initial denaturation at 94°C (10min) // followed by 40 cycles of denaturation at 94°C (12s), annealing at 59°C (30s), extension at 70°C (25s) // final extension of 72°C (7min)| 

PCRs were conducted using **10× dilutions of the dissolved material**, applying the following PCR recipe: 
* 11μl of mastermix from CWBio 
* 2μl of 1mg/ml BSA
* 2μl of 10μM of each primer
* 5μl of DNA 

--> Three independent PCR replicates were performed for each sample, each of the three replicates were run in separate PCR plates with each plate containing a negative.

## Sequencing
* Nine independent amplification-free DNA libraries were prepared for Illumina sequencing --> Libraries were prepared using VAHTS Universal Pro DNA Library prep kit for Illumina ND608.
* Each library consisted of samples from a single PCR replicate and three libraries each were prepared per primer pair. 

>Library preparation and sequencing was outsourced to GeneWiz (From Asenta Life Science) using Illumina NovaSeq to obtain:
>* 250 × 2 bp sequences for the 313-bp COI amplicon
>* 150 × 2 bp sequences for the 244-bp COI amplicon and 16S amplicons



In order to assess the performance of nanopore sequencing, we also sequenced one PCR replicate for the amplicon pools of all three primer pairs with MinION. 
>The DNA library was prepared using SQK-LSK110 ligation sequencing kit (Oxford Nanopore sequencing) for the 313-bp COI amplicon pool and the SQK-LSK112 kit for the 244-bp COI and 93-bp 16S amplicon pools, using recommended protocols with the modifications as follows: we excluded the FFPE DNA repair mix in the end-repair reaction and instead the reaction consisted of 50μl of DNA, 7μl of Ultra II End-prep reaction buffer (New England Biolabs), 3μl of Ultra II End Prep enzyme mix (New England Biolabs). An additional modification was the use of 1X ratio of Ampure XP beads accounting for the short fragment lengths sequenced in this study. 

Sequencing was conducted on three separate flowcells for each amplicon pool:
* 313-bp COI: R10.3 flowcell, MinION Mk1B; 
* 244-bp COI: R10.4 flowcell, MinION Mk1B; 
* 93-bp 16S: R10.4 flowcell, MinION Mk1C.

**Obs:** The high read counts for negatives of 244-bp COI fragment is due to coamplification of bacterial DNA by this primer.



## DNA barcodes to characterize the fly community
**Extraction:** Fly legs were removed and DNA extracted using 10–15 μl of HotSHOT (Truett et al., 2000). 

**PCR amplification:** 658-bp COI barcode using HCO2198 and LCO1490 primers (Folmer et al., 1994) using protocols described in Srivathsan et al. (2021). The primers were tagged with 13-bp sequences suitable for MinION sequencing. 

**Purification:** The PCR products were pooled in equal volumes and purified using Ampure XP beads (Beckman Coulter). 

**Sequencing:** By applying the MinION based barcoding pipeline described in Srivathsan et al. (2021).
For MinION based barcoding, a library was prepared using SQK-LSK110 ligation sequencing kit and the products were sequenced in a used MinION R9.4.1 flowcell. Basecalling was conducted using Guppy under the super accuracy model. The resulting sequences were processed using ONTbarcoder (version 0.9.1) (Srivathsan et al., 2021) in order to obtain DNA barcodes using 200× coverage as threshold. 

The resulting barcodes were matched with GBIF's sequence-id engine (https://www.gbif.org/tools/sequence-id) in order to identify the insects. All identifications with ≥97% sequence identity were accepted at species level. Afterwards, the sequences were aligned using MAFFT version 7 (Katoh & Standley, 2013) and clustered at 3% using objective clustering in order to determine species diversity and abundance (Meier et al., 2006, https://github.com/asrivathsan/obj_cluster). For four unidentified clusters, genus level identifications were made based on morphology.

------
# 2023
:::info
:page_facing_up:	[Danabalan et al. (2023)](https://onlinelibrary.wiley.com/doi/full/10.1002/edn3.398)
:::

## DNA Extraction

Samples were sorted as being:
* bloodfed (BF)
* non-bloodfed (NBF)

DNA was extracted using a Stool DNA kit (Roboklon GmBH, Berlin, Germany), according to the manufacturer's instructions with some adjustments.


## DNA Amplification

Two short mitochondrial markers (mtDNA):

**NBF** -----------------------------------------------------------------------

| Target| Primers| PCR conditions |
| --- |--- | ---- |
|108-bp fragment of the 12 s rRNA gene - vertebrate DNA| 12 S-V5 (Riaz et al., 2011) |   UNG digest 45°C 7 min, 95°C 10 min; PCR cycle 95°C 5 min, 42 cycles (95°C 30 s, 60°C 30 s, 72°C 1 min), 72°C 10 min|
|94-bp fragment of the 16 S rRNA gene - mammal DNA |16Smam (Boessenkool et al., 2012) following the method described in Hoffman et al. (2018)|45°C 7 min, 95°C 10 min, 42 cycles (95°C 30 s, 64°C 30 s, 72°C 1 min), 72°C 10 min.|

Illumina adapters were added in a second PCR, cleaned (Agencourt® AMPure XP PCR purification, Beckman Coulter, California) and indexes added in a third short amplification round.

**Riaz et al. (2011):**
![](https://hackmd.io/_uploads/HkxbREyJa.png)

**Boessenkool et al. (2012):**
![](https://hackmd.io/_uploads/r1JKlHy16.png)

**Hoffman et al. (2018):**
![](https://hackmd.io/_uploads/S12PMS1Jp.png)

![](https://hackmd.io/_uploads/BkpKGBykp.png)



**BF** -----------------------------------------------------------------------
| Target| Primers| PCR conditions |
| --- |--- | ---- |
|12 S (340 bp)| Mam-12 S_340 (Kocher et al.,2017) |   UNG digest 45°C 7 min, 95°C 10 min; PCR cycle 95°C 5 min, 42 cycles (95°C 30 s, 60°C 30 s, 72°C 1 min), 72°C 10 min|
|94-bp fragment of the 16 S rRNA gene - mammal DNA |16Smam (Boessenkool et al., 2012) following the method described in Hoffman et al. (2018)|45°C 7 min, 95°C 10 min, 42 cycles (95°C 30 s, 64°C 30 s, 72°C 1 min), 72°C 10 min.|

A single amplicon was generated for each marker. These were cleaned using Agencourt® AMPure XP PCR purification (Beckman Coulter, California, USA). Cleaned products were then sanger sequenced (SMB Services, Berlin, Germany).

**Insects** --------------------------------------------------------------------
For the purpose of building a DNA reference library, insects were identified using the 658-bp COI barcoding fragment (Folmer et al., 1994) --> Cycling conditions: 95°C 5 min, 35 cycles (95°C 30 s, 50°C 30 s, 72°C 1 min), 72°C 10 min

---
To reduce the amount of human and pig contaminants that could be present in the fly samples due to the urban environment and outside eating areas, both human and pig blocking primers were used (Hoffman et al., 2018)

Additionally, all PCR mixes contained 0.3 U Amperase® uracil N-glycosylase (UNG; Invitrogen, Carlsbad, CA) and digestion with UNG was carried out to degrade potential contaminant PCR products prior to the start of the PCR cycle. 

As such PCR products were generated using 0.2 mM of dN(U) TP. Briefly, two amplicons per sample were generated and samples with both amplicons present were cleaned (Purelink gel extraction,
Invitrogen) and pooled.

:::info
:page_facing_up:	[Saranholi et al. (2023)](https://onlinelibrary.wiley.com/doi/abs/10.1111/1755-0998.13851)
:::

## Sampling
* Insects were collected in the Parque Ecológico de São Carlos (PESC), Brazil, a zoo that houses about 35 enclosed native and non-native mammal species
* Mosquitoes and flies were collected using CDC (Center for Diseases Control) light traps and fish-baited plastic bottle traps, respectively. 
* Insect collections were carried out for 5 consecutive days in the winter (June 2020), when the zoo was closed to the public due to the coronavirus pandemic condition. Eight sampling points were distributed across the PESC area (minimum distance of 100 m and maximum distance of 300 m among traps), totalling 40 insect-trap days
* In both types of insect traps, a 50 mL sterilized plastic tube containing absolute ethanol with a high purity level for molecular biology use was fitted at the bottom for immediate preservation of the collected insects. Each tube was replaced every day and the insects collected were kept in absolute ethanol at −20°C until their sorting and DNA extraction

## DNA Extraction
* From all insects collected, only female blood-fed mosquitoes and hematophagous and saprophagous flies were sorted out for DNA extraction. Mosquitoes were identified at the genus level, while flies were identified at the family level (both mosquito and fly identification were conducted by a specialist).
* Once insects were identified, the ethanol was drained, and the specimens were placed in a 37°C oven for a maximum of 4 h to guarantee complete evaporation of ethanol before DNA extraction.
* DNA extraction was performed separately for each individual collected using the Chelex protocol, following Casquet et al. (2012).
*  Before PCR, the DNA from individual insects were pooled in insect bulks, according to insect groups and sampling points

## DNA Amplification
* The mini-barcode sequences for the 12SrRNA and 16SrRNA mitochondrial (mtDNA) ribosomal genes were amplified using primers to target vertebrate (12SV5F and 12SV5R; Riaz et al., 2011) and mammal (16Smam1 and 16Smam2; Taylor, 1996) species, respectively. For the 12SV5F primer, the first nucleotide was changed to a degenerate base (5′-YAGAACAGGCTCCTCTAG-3′) to allow binding in more mammal species, as suggested by Kocher et al. (2017).
* To check for contamination, PCR amplifications included a non-template sample as negative control. 
* The amplified products were visualized on 1.5% agarose gels by electrophoresis. 
* A second PCR was performed for samples that failed in the first one, as a new attempt for amplification. With these steps, we successfully amplified the iDNA from all bulks of sorted insects