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The diets of fish are routinely examined by direction and research bureaus to turn to a broad array of subjects. The aims of these probes can be every bit simple as set uping an stock list of what prey points are consumed or every bit complex as mensurating dietetic responses to alterations in the environment ( Bowen 1992 ) . Historically, the criterion protocol for depicting the diet involves visually placing the contents of fish tummies and sum uping prey points by figure, frequence of happening, and either volume or weight ( Hyslop 1980 ) . Despite widespread usage, there are restrictions built-in to ocular techniques that can bias consequences. For illustration, the rate at which different types of quarry digest in the tummy varies, and as a consequence some quarry points will be noticeable for longer clip periods than others ( Gannon 1976 ; Berens and Murie 2008 ) . Besides, physical constructions of the fish can powderize certain prey points, doing designation hard if non impossible.

Options to ocular analysis of tummy contents are available, and Polymerase Chain Reaction ( PCR ) techniques represent an appealing option. PCR uses a series of chemical reactions to bring forth 1000000s of transcripts of targeted Deoxyribonucleic acid fragments that can unambiguously place to single species ( Hebert et al. 2003 ) . Merely proceedingss sums of tissue recovered from quarry would be needed for PCR due to the exponential nature of the reaction. PCR is a widespread tool used in a diverse array of research Fieldss and as consequence databases have been created that contain 100s of 1000s of DNA sequences from known persons ( e.g. GenBank, hypertext transfer protocol: //www.ncbi.nlm.nih.gov/Genbank/ ) . The public-service corporation of these expansive databases is that they allow for used for comparing and subsequent designation of unknown samples through trials of sequence similarity. Finally, DNA based attacks have been used to place single quarry points with losing or degraded morphological features in birds, Marine mammals, and fish ( Jarman et al. 2004 ; Blankenship and Yayanos 2005 ; Smith et Al. 2005 ; Deagle et Al. 2005b ; Deagle 2006 )

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The overall end of this survey was to compare the consequences from two techniques, ocular and molecular analysis, applied to the same set of prey points recovered from the tummy contents of Gallic oink ( Haemulon flavolineatum ) . The ensuing prey designation determined by each method entirely and combined was compared. This research examined the potency for molecular analysis to place samples that would be unidentifiable by ocular analysis entirely.

Ocular analysis of tummy contents was detailed in Chapter 2, and trials were conducted to look for alterations in diet by trying event, location and fish size. These ends were addressed through the quantification of prey points and comparing of niche comprehensiveness and dietetic convergence ( Chapter 2 ) . Molecular designation of prey points was performed to find if prey type, prey province of digestion, or marker choice affected the ability to sequence extracted DNA ( Chapter 3 ) . Finally, critical factors act uponing the success of both ocular and molecular analysis from the same samples will be discussed ( Chapter 4 ) .

Chapter 2

Food wonts of Gallic oink

Introduction

Gallic oink occur in Waterss less than 60 metres deep in the western Atlantic from South Carolina to Brazil, including parts of the Gulf of Mexico. Gallic oink are common throughout much of their scope and in some locations are the most abundant fish species observed on coral reefs ( Randall 1967 ) .

Gallic oink undergo ontogenetic displacements in home ground usage, switching from nursery countries to coral reefs ( Cocheret de la Morini & A ; egrave ; re et Al. 2003a ; Cocheret de la Morini & A ; egrave ; re et Al. 2003b ; Nagelkerken and van der Velde 2004 ; Nagelkerken and Velde 2004 ) . Newly settled larvae inhabit interstitial infinites in seagrass beds and occur as lone persons. Juveniles aggregate into schools and use mangrove roots, spot reefs, and constructions within seagrass beds as baby’s room home ground ( Cocheret de la Morini & A ; egrave ; re et Al. 2003a ; Cocheret de la Morini & A ; egrave ; re et Al. 2003b ) . As persons reach sexual adulthood, they form resting schools on coral reefs and finally travel to offshore home grounds ( Meyer and Schultz 1985 ) .

Gallic oink provender via a sifting behaviour, whereby possible quarry and non-nutritive dust are separated within the oropharyngeal pit. Prey points that are retained are macerated by pharyngeal dentitions before come ining the tummy. Several surveies have documented the diet throughout their scope, including the Netherland Antilles ( Cocheret de la Morini & A ; egrave ; re et Al. 2003a ; Cocheret de la Morini & A ; egrave ; re et Al. 2003b ; Nagelkerken and van der Velde 2004 ; Nagelkerken and Velde 2004 ) , Puerto Rico ( Austin and Austin 1971 ; Dennis 1992 ) , Haiti ( Beebe and Tee-Van 1928 ) , Florida ( Davis 1967 ; Hein 1996 ) and the United States Virgin Islands ( Randall 1967 ) . Diets change as fish passage through home grounds, and major scrounging clubs have been determined by size: pre-juveniles ( & A ; lt ; 50 millimeter FL ) , juveniles ( 51-150 millimeter FL ) and grownups ( & A ; gt ; 150 millimeter FL ) ( Hein 1999 ) . Pre-juvenile fish provender during the twenty-four hours on planktonic copepods within the baby’s room home ground ( Gaut and Munro 1983 ; Cocheret de la Morini & A ; egrave ; re et Al. 2003a ) . Juveniles get down doing migrations to seagrass beds to scrounge at dark on benthic invertebrates which includes tanaids, decapod pediculosis pubiss and runt, and polychaete worms ( Dennis 1992 ) . Adults form resting schools on coral reefs and do crepuscular migrations to grassbeds and sand spots where they consume chiefly polychaete and sipunculid worms, univalves and decapod crustaceans ( Randall 1967 ; Estrada 1986 ; Dennis 1992 ) .

The comparative importance of prey points varied some between surveies ; nevertheless, unidentifiable quarry points were routinely encountered. Dennis ( 1992 ) recorded at least one unidentified quarry point in 263 out of 330 ( 79 % ) tummy examined, and Cocheret de la Moriniere et Al. ( 2003a ) found unidentified stuff constituted up to 58 % of entire tummy contents by volume for peculiar sized fish.

Gallic oink were selected as the survey species for two grounds: 1 ) their ecological importance within the seagrass and coral reef home grounds as marauders of benthal invertebrates and transporters of foods between home ground types, and 2 ) in order to qualitatively heighten diet consequences that have often contained big Numberss of unidentified quarry.

The aims of this chapter were to visually quantify the diet of Gallic oink, and trial for alterations in diet by trying event and home ground type. Frequency of happening informations for assorted prey types from this chapter will be used for direct comparing with the consequences of molecular analysis of tummy contents in chapter 4.

Methods

Gallic Grunt Collections

Gallic oink were collected from the southern shore of St. John Island, US Virgin Islands, USA, in June of 2008 and May/June of 2009. For each person, the clip of twenty-four hours, day of the month, GPS location, fork length ( FL ) , deepness ( metres ) , and gear type was recorded. In 2008, aggregations were made throughout the twenty-four hours to find periods of extremum forage and one time established sampling was targeted towards times with greatest opportunity of quarry filled tummies. In entire, fish were collected from two reef home grounds ( Tektite and Fish Bay ) and one seagrass home ground ( Viers Dock ) .

Grunts were collected utilizing multiple cogwheels including hook and line, trap, pole lance, and manus cyberspace. Hook and line trying consisted of sabiki rigs with multiple maulerss baited with calamari. Fish traps ( 1m long, 1m broad, and.5m in tallness ) with mesh size of ~7.5cm2 were set on sand spots near reef home grounds, baited utilizing cat nutrient ( Kozy Kitten ) , and set overnight. Both spear fishing and manus cyberspace aggregations were conducted by frogmans utilizing scuba cogwheel. Collections by manus cyberspace utilized a modified dramatis personae cyberspace ( brail and manus lines removed ) to capture Gallic oink.

Fish captured alive were brought to the surface and their and tummy contents were obtained via stomachic lavage ( Foster 1977 ; Light et Al. 1983 ; Hartleb and Moring 1995 ) . A 3 millimeter diameter tubing attached to a pressurized sprayer armored combat vehicle was inserted into the fish ‘s gorge and H2O was pumped in pulsations as the wand was moved back and Forth to loosen prey points. Regurgitated contents were deposited onto a 200 micrometer screen and tummy contents were collected and put into separately labeled unfertile trying bag. Sampling bags were kept on ice in the field ice prior to stop deading in the lab. For fish less than 75 millimeters FL a 10 milliliter panpipes rigged with an blow uping acerate leaf were substituted in the topographic point of the sprayer armored combat vehicle. Fish harvested utilizing a pole lance were placed straight into single plastic bags underwater and placed on ice at the surface prior to transit to VIERS research lab. Speared oinks were dissected, and contents from the gorge to upper bowel were removed for ocular analysis.

Stomach Content Analysis

Stomach contents were transported to University of Florida FAS installations where they were thawed and examined utilizing a Leica MZ 12.5 stereomicroscope. Diet points were identified to the lowest systematic degree utilizing relevant designation keys ( Maning 1969 ; Fauchald 1977 ; Abele and Kim 1989 ; Kensley and Shotte 1989 ; Thomas 1993 ; Cutler 1994 ; Hendler et Al. 1995 ; Heard et al. 2003 ) . Individual prey points were catalogued by fish figure, given a distinguishable designation figure and photographed utilizing a digital imagination system ( Motic ) . Once sorted, single points were rinsed with deionized H2O and placed into single 1.5 milliliter Eppendorf phials incorporating 100 % non-denatured ethyl alcohol.

Recovered nutrient points were catalogued by per centum happening ( % O ) , and per centum by figure ( % N ) . Percent happening is defined as the entire figure of tummy incorporating a peculiar quarry type divided by the entire figure of stomachs incorporating nutrient. Numeric copiousness is calculated as the figure of each quarry type from all tummies divided by the figure of all prey points from all tummies. Volumetric measurings were non taken because prey types were diverse and capable to volumetric deformation as a consequence of digestion. Hydrometric measurings were non taken for two grounds: 1 ) due to the mistake associated with wet weights and the inability to take H2O every bit from all prey types and 2 ) to minimise processing clip between dissolving and submergence into DNA preservative.

Accumulative quarry curves, which count the figure of new prey point per each person tummy, were generated to measure the adequateness of sample size ( Ferry and Caillet 1996 ) . A random figure generator was used to find the order in which tummy were analyzed and the figure of fresh prey points per new tummy was recorded. This procedure was iterated 10 times to set up norms and standard divergences for each tummy figure. A graph demoing the entire figure of stomachs versus the figure of new prey points in each tummy was generated. If the curve for this graph reaches an asymptote so the diet is considered to be good described ( Ferry and Caillet 1996 ) .

Niche Breadth and Diet Overlap

Comparisons between trying events and home ground types were calculated utilizing % N. Niche comprehensiveness was calculated utilizing Levin ‘s standardised index ( BA ) to find if differences in trying locations and aggregation event influenced the niches exploited ( Krebs 1999a ) . This index returns ranges on a graduated table from 0 ( narrow niche ) to 1 ( wide niche ) .

Diet convergence for trying event, home ground type and fish size was calculated utilizing Morisita ‘s index of similarity. Morisita ‘s index was selected because of low prejudice with varied samples sizes and big Numberss of resource provinces ( Smith and Zaret 1982 ) . Both sets of computations were performed utilizing Ecological Methodology package bundle ( Krebs 1999b ) .

Consequences

Gallic Grunt Collections

A sum of 99 fish were collected during two trying trips in June of 2008 ( 69 specimens ) and May/June of 2009 ( 30 specimens ) . Multiple gear types were employed in both old ages with the bulk of fish collected utilizing a manus cyberspace ( Table 2-1 ) . Overall, sampled Gallic oink ranged in size from 57-188 millimeter FL ( Figure 2-1 ) with a average length of 119 millimeter ( SD = 40.3 ) . Fish collected in 2008 spanned a larger scope of sizes and displayed a bimodal distribution with extremums around 70 centimeter and 170 centimeter. Samples from 2009 were significantly larger on norm than 2008 ( Welch ‘s t-test=-2.92, df=76.5, p=0.004 ) and were distributed equally across sizes from 80 millimeters to 180 millimeter. Multiple habitat types were sampled with 69 ( 70 % ) fish collected from coral reefs and 30 ( 30 % ) from seagrass beds. Fish collected on coral reefs were larger in general compared to seagrass beds and encompassed a larger scope of sizes.

Samples collected at different times during the twenty-four hours in 2008 indicated forenoon hours yield more tummies filled with quarry and later all aggregations in 2009 occurred prior to 09:00 AM ( GMT- 4:00 ) ( Figure 2-2 ) .

Stomach Content Analysis

In 2008 a sum of 69 fish were collected and 26 ( 38 % ) of tummy contained prey points. From 2009, a sum of 30 fish were collected and 25 ( 83 % ) had tummy contents. Regardless of digestion codification, all prey points recovered were included in analysis.

Gallic oink collected 2009 contained more prey points on norm ( 2.6 in 2008 vs. 7 in 2009 ) and a greater array of quarry types. Unidentified crustaceans were normally encountered in both old ages by happening ( 46 % and 48 % ) and figure ( 13 % and 10 % ) for 2008 and 2009 severally ( Table 2-2 ) . Polychaete worms were abundant by happening ( 19 % and 20 % ) as were harpacticoid copepods ( 23 % and 16 % ) once more for 2008 and 2009 severally. Numerically unidentified quarry points ( 6 % and 13 % ) and polychaete worms ( 5 % and 3 % ) were normally counted prey points. Sipunculid worms showed the largest difference between old ages by happening ( 15 % and 96 % ) and figure ( 8 % and 35 % ) for 2008 and 2009 samples severally. Unidentified prey points were less common by happening in samples from 2008 ( 23 % ) relation to 2009 ( 68 % ) . Stomatopods and taniad crustaceans were absent from tummy collected in 2008 but present in 2009 samples ( 36 % and 16 % by happening and 7 % and 2 % numerically ) .

Diet by Sampling Location

Stomach contents of Gallic oink were divided by trying location to analyze possible tendencies. In entire, two coral reef home ground types were sampled, Fish bay ( n=9 tummy ) and Tektite reef ( n=27 tummy ) , and one seagrass bed, Viers Dock ( n=15 tummy ) . Prey types were lumped into broader systematic classs ( Amphipoda, Copepoda, etc. ) to simplify analysis. Several similarities were observed between diets collected at the three sites. Unidentified crustaceans were abundant by % O ( 53 % , 56 % , and 37 % ) and % N ( 17 % , 15 % , 10 % ) for Viers Dock, Fish Bay, and Tektite Reef severally ( Table 2-3 ) . Unidentified prey points were besides abundant at all musca volitanss ( 33 % VD, 56 % FB, and 48 % TR ) by happening.

Distinct differences were revealed by location with fish merely being consumed at the Viers Dock location ( 13 % and 17 % ) by % O and % N. Foraminifera ( 4 % and 3 % ) and Polyplacophora ( 4 % and 1 % ) by % O and % N were merely observed in tummy from Tektite reef. Stomatopods ( 11 % for both locations by % O ) and tanaid crustacean ( 11 % FB and 30 % TR ) were identified merely from reef samples. Sipunculid worms were consumed at all three locations, with more persons consumed by happening and figure in Fish Bay ( 89 % O and 32 % N ) and Tektite reef ( 67 % O and 35 % N ) relative to Viers Dock ( 13 % O and 13 % N ) .

Diet by Size

The diet of Gallic oink was divided into two size groups ( A = & A ; lt ; 90mm criterion length ( SL ) and B = & A ; gt ; 90mm ) to analyze possible forms in the copiousness and frequence of different prey points. These size groups were selected because old work has suggested that nocturnal forage begins during a mid-juvenile stage ( Hein 1999 ) . Fork lengths of Gallic oink collected in this survey were converted to standard length ( SL ) utilizing the arrested development FL = 1.04 * SL + 4.04 ( Hein 1992 ) . Prey types were lumped into broader systematic classs ( Amphipoda, Copepoda, etc. ) to analyze general tendencies. In entire, size group A contained 25 persons and group B had 26 persons.

For club A the most of import quarry points numerically were unidentified crustaceans ( 18 % ) , sipunculid worms ( 17 % ) , copepods ( 10 % ) , fish ( 9 % ) and polychaetes ( 8 % ) ( Table 2-4 ) . By happening, unidentified crustaceans besides dominated ( 52 % ) followed by copepods ( 28 % ) , polychaete worms ( 24 % ) , runts ( 24 % ) , and peanut worms ( 24 % ) . For club B, sipunculid worms were most of import by figure ( 36 % ) , unidentified quarry points ( 15 % ) , unidentified crustaceans ( 10 % ) and tanaid crustaceans ( 6 % ) . By happening, sipunculid worms were consumed by about all persons ( 85 % ) , followed by unidentified quarry types ( 69 % ) , unidentified crustaceans ( 42 % ) and ophiuroids ( 27 % ) . Both runt and pediculosis pubiss were more normally consumed by smaller persons than the larger groups ( 44 % O and 23 % O ) .

Accumulative Prey Curves

The cumulative quarry curve constructed based on all diet points consumed by all fish collected did non make an asymptote, bespeaking that the diets of Gallic oink were non good characterized.

Niche Breadth and Diet Overlap

Niche comprehensiveness for the two trying trips was well different ( 0.19 and 0.52 for 2008 and 2009 severally ) . The figure of resource provinces utilized was higher in 2009 ( 38 ) compared to 2008 ( 18 ) , nevertheless fish from 2009 used merely three resources often. Niche overlap as calculated utilizing Morisita ‘s simplified index for the two trying trips was moderate ( 0.62 ) .

When analyzed by location, niche comprehensiveness was greatest at Viers dock ( 0.64 ) followed by Fish Bay ( 0.41 ) and Tektite Reef ( 0.31 ) . The figure of resource provinces was similar amongst sites ( VD=13, FB=14, TR =17 ) with 8 resources normally used at Viers Dock and Fish Bay and merely 5 resources on Tektite Reef. Niche convergence was higher between Fish Bay and Tektite Reef ( 0.91 ) compared with Fish Bay to Viers Dock ( 0.70 ) or Tektite Reef to Viers Dock ( 0.63 ) .

By size, the niche comprehensiveness was greater for smaller persons ( 0.64 ) relation to larger 1s ( 0.27 ) as calculated by Levin ‘s standardised step. Both clubs utilized an array of quarry types ( Guild A = 14, B=18 ) , nevertheless much fewer prey classs were routinely utilized by larger persons ( 4 vs. 8 for A and B severally ) . A moderate sum of diet convergence between clubs was observed harmonizing to Morisita ‘s simplified index of convergence ( 0.73 ) .

Discussion

Sipunculid worms were both numerically and by happening the most normally consumed prey point by Gallic oinks collected in this survey. The bulk of sipunculid worms counted in this analysis were partial beings and consisted of little more than the distal part of the sipunculid introvert, proposing that numerically their importance might be overestimated. Both unidentified crustaceans and unidentified quarry points were found in about half of all persons stomachs analyzed and represented of import quarry types numerically. Prey points coded as unidentified crustaceans or unidentified quarry encompassed a broad scope of sizes and without hydrometric or volumetric measurings minimum illation can be made sing the dietetic importance of theses prey types. Harpacticoid copepods were found in a big part of the tummy nevertheless probably contributed small nutritionary value to the diet of Gallic oink given their little size ( 1mm sum length ) .

When tummies were divided by trying location sipunculid worms were found to be of import numerically and by happening in both reef home ground types, but were consumed much less often from the seagrass home ground. There is no clear account why sipunculid worms might be consumed more normally on coral reefs without cognizing something about the comparative copiousness of prey points. Stomatopods and tanaid crustaceans were merely observed in tummy collected on reefs and once more this might be related to feed copiousness.

Stomach contents of persons categorized by size revealed that fish less than 90mm SL was the lone size category to devour fish as quarry and can potentially be explained by the big schools of silversides ( Atheriniformes ) nowadays in the sampling country. Fish greater than 90mm SL consumed the lone ophiuroids recovered from tummy contents nevertheless none of these persons represented whole persons but distal parts of legs.

When calculated for all persons, niche comprehensiveness suggests that Gallic oink work neither a narrow nor wide niche as evidenced by moderate Levin ‘s index of niche comprehensiveness ( 0.38 ) . Niche comprehensiveness appeared to diminish with fish size proposing that either fewer quarry types are exploited or that diets are to a great extent influenced by specific quarry types consumed in big measures. Similarity between diets was greatest between the two reef trying sites and to a lesser extent seagrass home ground harmonizing to Morisita ‘s index of similarity which is what one would anticipate.

The consequences of this survey are by and large consistent with old diet surveies conducted in the part, nevertheless the ability to pull direct comparings to old surveies was limited due to different indices used to catalog prey points. Dennis ( 1992 ) analyzed the diets of 330 Gallic oink from Puerto Rico and concluded that polychaete worms, peanut worms, univalves and runt were the four most of import quarry types by biomass. Randall ( 1967 ) found that for Gallic oinks collected in the US Virgin Islands and Puerto Rico, polychetes were the most of import quarry type volumetrically followed by pediculosis pubis and sipunculid worms. Estrada ( 1986 ) examined Gallic oink diets from Columbia and concluded that crustaceans, molluscs and polychetes were most normally encountered by frequence and consumed more worms ( peanut worms and polychetes ) relative to other oink species. Interestingly plenty, multiple surveies conducted in the Netherland Antilles concluded that neither sipunculid worms nor polychetes were ingested at any degree ( Cocheret de la Morini & A ; egrave ; re et Al. 2003a ; Cocheret de la Morini & A ; egrave ; re et Al. 2003b ; Nagelkerken and van der Velde 2004 ) . Diets from fish collected on coral reefs were dominated volumetrically by decapods pediculosis pubiss and prey fishes while fish from nursery home grounds consumed chiefly tanaid crustaceans, copepods and decapods crustaceans ( Cocheret de la Morini & A ; egrave ; re et Al. 2003a ; Cocheret de la Morini & A ; egrave ; re et Al. 2003b ) .

The high rate of tummy contents with unidentified quarry points and the fact that old surveies produced similar consequences provides justification for researching alternate agencies of designation, specifically molecular analysis which does non trust on morphological features to bring forth designations.

Table 2-1. Gallic oink aggregations by twelvemonth and gear type.

Table 2-2. Occurrence ( % O ) and numerical copiousness ( % N ) for quarry sampled from Gallic oink tummy contents collected from St John, US Virgin Islands in May/June of 2008 and June of 2009.

Table 2-3. Occurrence ( % O ) and numerical copiousness ( % N ) for Gallic oink tummy contents divided by trying site.

Table 2-4. Diet of French oink by figure ( N % ) and ( O % ) based on size class.

Figure 2-1. Size distribution of Gallic collected from St. John, US Virgin Islands in May/June of 2008 and June 2009.

Figure 2-2. Collection times and figure of persons incorporating tummy contents by clip for Gallic oinks.

Figure 2-3. Accumulative quarry curve for all prey points recovered from Gallic oinks collected in 2008 and 2009.

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