Exploration for natural enemies of Hydrilla verticillata in East/Central Africa and genetic characterization of worldwide populations




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Exploration for natural enemies of Hydrilla verticillata in East/Central Africa

and genetic characterization of worldwide populations.
W. A. Overholt1, R. Copeland2, D. Williams3, J. Cuda4, B. Nzigidahera5, E. Nkubaye5, F. Wanda6 and B. Gidudu6
1 Biological Control Research and Containment Laboratory, University of Florida, Fort Pierce, FL

2 International Center of Insect Physiology and Ecology, Jinja, Uganda

3 Department of Biology, Texas Christian University

4 Department of Entomology and Nematology, University of Florida, Gainesville, FL

5 Institute National pour l’Environnement et la Conservation de la Nature, Bujumbura, Burundi

6 National Fisheries Resources Research Institute, Jinja, Uganda
Abstract
Surveys in Burundi and Uganda indicate that chironomid midges comprise the great majority of insects associated with Hydrilla. In total, 21,817 chironomids, representing 17 species have been reared from collections made in Lake Tanganyika, Burundi, with Dicrotendipes fusconotatus accounting for 84% of individuals. The putative Hydrilla herbivores, Polypedilum dewulfi and Polypedilum wittei, were reared in relatively large number from two locations near Bujumbura in Lake Tanganyika, and efforts to colonize these insects have been initiated. In Uganda, a total of 1886 chironomids representing 35 species were reared from Hydrilla, 20 of which were collected in Lake Bisina, the most frequently sampled lake. Dicrotendipes septemmaculatus, a close relative of D. fusconotatus, accounted for 27% of specimens. Neither of the two target Polypedilum spp. was commonly reared in Uganda. Berlese funnels set up in Burundi recovered chironomids, but only one curculionid larva. One pyralid larva was collected from Hydrilla in Lake Tanganyika on a leaf whose margin had been partially chewed. No insects were reared from over 100 Hydrilla tubers collected at Cercle Nautique, Lake Tanganyika.
A plant diversity survey in Lake Bisina, Uganda found 10 plant species, with 9 submersed macrophytes and Nymphea nuchalli. Najas horrida was the most widespread and abundant species. Hydrilla was found at about one-half of sampled sites and was the fourth most abundant plant. Hydrilla was typically found growing in communities with 3-4 other plants species.
DNA has been extracted from a total of 488 Hydrilla samples. Seventeen polymorphic Hydrilla microsatellite loci have also been isolated and dye labeled. We expect at least 10 of these loci will provide easily interpretable genotypes. We successfully amplified a portion of the pds gene using nuclear DNA as judged by its perfect sequence alignment to the published cDNA sequence of the pds gene. We are currently testing this region’s utility as a marker for resistance using fluridone resistant strains of Hydrilla.
Introduction
Classical biological control of weeds involves exploration for a weed’s natural enemies in its native range. The native range of Hydrilla includes much or Asia and northern Australia. Populations have also been reported at a few locations in Europe and in East/Central Africa (Cook and Löünd 1982). The African populations were thought to be native as Hydrilla was found in the region as early as the 1860s. However, genetic studies conducted in 2006/2007 by the current project revealed that the African populations have very low diversity and closely match the genetic makeup of Hydrilla from India and the southern United States, suggesting that the African populations are the result of an early introduction. In comparison, genetic diversity in China was very high. The distribution of Hydrilla in East/Central Africa also supports an introduction hypothesis as Hydrilla has only been found 5 of 21 lakes surveyed by the project. If Hydrilla was native to this part of Africa, it would be expected to have a less restricted distribution.
Despite the growing evidence that Hydrilla in East/Central Africa is a result of an introduction, the plant does not have invasive characteristics in the lakes where we have found it. It does not form large monocultures and only very rarely reaches the surface. Additionally, it is not considered to be problem in any of the locations where it occurs in East/Central Africa. Thus, it appears that biotic and/or abiotic factors maintain Hydrilla at low density. Early surveys in the region (Pemberton 1980, Markham 1985) identified two chironomid midges as Hydrilla herbivores (Buckingham 1994); Polypedilum dewulfi and Polypedilum wettei, but efforts to colonize these insects failed (Bennett and Buckingham 2000). Both Pemberton and Markham also suggested that fish may feed on Hydrilla.
Genetic studies can be very useful in determining the native range of organisms and to pinpoint the likely origin of introduced populations. Earlier studies using randomly amplified polymorphic DNA suggested that southern India was the source of Florida dioecious Hydrilla but did not speculate as to a center of origin for the species (Madeira et al. 1997). The highest diversity of natural enemies is hypothesized to occur in the area where an organism has occurred for the longest period (Van den Bosch et al. 1982). The highest genetic diversity of Hydrilla has been found in China (June 2007 Project Report), with very low diversity, but similar genetic types in Africa, India and the southern USA. An Asian center of origin is supported by the USDA/ARS exploration efforts in Asia where a much higher diversity of natural enemies is being found than in Africa (USDA/ARS Hydrilla Project Reports, Wheeler, personnal communication).
The main objective of the current project is to determine whether insect herbivores are involved in the regulation of Hydrilla in East/Central Africa, and if so, to colonize the insects for host range studies. Additionally, genetic studies are being conducted to determine the relationships among worldwide populations of Hydrilla, and to develop a better method of assaying for Hydrilla resistance.
Methods and Materials
Surveys in Uganda and Burundi: Because of the absence of Hydrilla from in Kenyan (June 2007 Project Report), Copeland moved to Jinja, Uganda on 1 March 2008. He had intended to move on 1 January 2008 but was prevented from traveling there by road, due to the civil unrest following the elections in Kenya in December 2007. He made a trip by air to Uganda in Feb 2008 and travelled to Burundi for week-long field trips in February, April, and late May. Fiber optic illuminators were brought to each country in February, and these have greatly improved conditions in the laboratories.
The general methology of surveys has been to collect large amounts of Hydrilla by wading and hand-pulling or using grapple hooks from boats. The Hydrilla is then transported to laboratories in Jinja (Uganda) or Bujumbura (Burundi) in closed 40 liter plastic containers with water from the collection site. Jerry cans of water from each collection site are also collected and transported to laboratories along with the Hydrilla. At the laboratories, Hydrilla is carefully separated from any other aquatic vegetation and placed in clean 40 liter plastic containers with water collected at the same site. The containers are covered with fine mesh netting, followed by a piece of black plastic, and sealed with a lid that has been fitted with a plastic funnel. An acrylic box for trapping emerging insects is placed over the funnel. Air is bubbled into the water in containers through a tube connected to a small aquarium pump. The water is changed about once a week to slow decomposition of the Hydrilla. After 2-3 weeks, the Hydrilla is removed and discarded.
The acrylic boxes are inspected daily and any insects that have emerged are collected in vials in 70% ethanol and labeled. Insects in vials are examined by Bob Copeland and then sent to Dr. John Epler (a leading aquatic insect taxonomist) for identification. We anticipate that over time, fewer insects new to Copeland will be found and he will be able to make most of the identifications in Africa.
Plant inspection: Terminal tips (4-5 cm) of individual plants (161) were collected in Lake Bisina, Uganda in May, 2008 and closely inspected under a microscope to look for insect herbivores and signs of herbivory.

Laboratory colonization of Polypedilum spp. Large colony cages have been erected in Burundi and Uganda in preparation for attempts to rear one of both of the putatively herbivorous Polymedilum spp. (P. dewulfi and P. wittei) Each cage contains a large aquarium for culturing Hydrilla. Cultures have been established in both countries with native Hydrilla. In Burundi one cage is set outdoors and the other indoors with artificial lighting. The cage in Uganda is set in a well lit room, with natural light.
Lake Bisina surveys: Four surveys were conducted in 2008; January, March, May and June. During each survey, a boat was used to follow transects dissecting the lake at different points (Figure 1). The boat stopped every 100-500 meters, and a grappling hook was tossed three times at different directions. Submersed plants were dragged back to the boat and inspected (Figure 2). Plant abundance was determined using a scale as follows: Dominant, Abundant, Frequent, Occasional, Rare or Absent. Water depth and clarity were also measured. Where Hydrilla was found, a sample was collected to rear out any arthropods. In addition, in the May survey, 100 Hydrilla tips (4-5 cm) were dissected to look for insect damage. Water quality (dissolved oxygen, pH, total dissolved solids, conductivity and Redox) was measured during the June survey. To date, 112 locations have been sampled.








Figure 1. Uganda map (left) indicating location of Lake Bisina and sampling points in Lake Bisina.

Figure 2. Collection of submersed plants from Lake Bisina, Uganda.
Genetic studies: DNA has been extracted from a total of 488 Hydrilla samples using a modified extraction method of Kim et al. (1997). Approximately 40% of the samples needed to be re-purified using a Promega Genomic DNA Wizard cleaning kit to further remove compounds that were inhibiting PCR amplifications. Most of the collected samples are from the USA (N = 329; 252 of which are from Florida) and East Africa (N = 50). The rest of the samples (N = 109) are from various areas in Asia and the Indo-Pacific region.

Microsatellite loci: Microsatellite loci were isolated using a modified protocol of Travis and Schable (2005). Briefly, Hydrilla DNA from Florida and East Africa was combined and then digested with restriction enzymes RsaI and AluI. The restricted DNA was then gel purified to further remove any potential inhibitory compounds that might have been present. Linkers were then attached to the ends of the digested DNA fragments and PCR was used to amplify the DNA fragments using primers specific to these linkers. We then hybridized these amplified fragments to cocktails of biotin-labeled microsatellite repeats [(ATC8, ACT12, GAA8, GA12) and (TGC8, CCA8)]. The hybridized fragments were then captured with streptavidin coated magnetic beads (which bind to the biotin ) (Promega). The resulting bead and fragment mixes were then washed to remove fragments that were not hybridized to a microsatellite repeat (2X at room temperature with 2X SSC, 2X at room temperature with 1X SSC, and 2X at 5oC below hybridization temperature with 1X SSC). The remaining “enriched” fragments were then PCR amplified again and ligated into pGEM-T vectors (Promega) , transformed into JM109 high-efficiency competent cells, and plated on LB/ampicillin plates. The resulting colonies were then put directly into PCR reactions with vector primers T7 and SP6 to amplify the cloned Hydrilla DNA fragments. The amplified clones were then sequenced in both directions with an ABI BigDye v3.1 cycle sequencing kit and electrophoresed on an ABI 3130 Genetic Analyzer (PE Biosystems). Sequences were edited and aligned using Sequencher v 4.8 (Gene Codes).
Screening for fluridone resistance: We used the published cDNA sequence of the phytoene desaturase (pds) gene (GenBank AY639658) to design primers to amplify a segment that is 200-300 base pairs in length and includes the mutation region conferring resistance to fluridone (Michel et al. 2004, Puri et al. 2007a). The amplified products were sequenced with an ABI BigDye v3.1 cycle sequencing kit and electrophoresed on an ABI 3130 Genetic Analyzer.
Results
Surveys in Uganda and Burundi: Throughout the project, Chironomidae comprised the great majority of insects reared from Hydrilla. In total, 21,817 chironomids, representing 17 species have been reared from collections made in Lake Tanganyika, Burundi (Table 1). One species, Dicrotendipes fusconotatus made up 84% of specimens. We were finally able to rear our target chironomid species, Polypedilum dewulfi and P. wittei in relatively large numbers. On separate sampling dates (3 Nov 2007, 20 Feb 2008) at two different sites (Saga Vodo and Cercle Nautique) near Bujumbura, 123 and 149 P. dewulfi were reared from Hydrilla. In addition, 86 P. wittei were reared from the 20 Feb 2008 Cercle Nautique Hydrilla sample.
In Uganda, a total of 1886 chironomids representing 35 species were reared (Table 2). Twenty species were recovered from Hydrilla collected in Lake Bisina, our most frequently sampled lake. Dicrotendipes septemmaculatus, a close relative of D. fusconotatus, accounted for 27% of specimens. Neither target species was commonly reared from Hydrilla growing in Uganda lakes. Only 19 specimens of P. wittei were reared, and only a single specimen of P. dewulfi.
Few other insects were reared from Hydrilla. With the exception of the trichopteran, Orthotrichia sanya Mosely, which was relatively common in Lake Tanganyika (but rare in Ugandan Lakes) non-Chironomidae were rarely recovered from Hydrilla. Another Trichoptera, Ecnomus thomasetti Mosely was reared both from Ugandan lakes and Lake Tanganyika, while Ecnomus kunenensis was rare and found only in Lake Bunyonyi in western Uganda. A single curculionid larva was recovered from Berlese funnel traps containing Hydrilla from Lake Tanganyika. Berlese traps produced mostly chironomid larvae along with a few adults. Chironomid species recovered from Berlese traps did not differ from those reared from Hydrilla held in water. One pyralid larva was collected from Lake Tanganyika Hydrilla on a leaf whose margin had been partially chewed away. No insects were reared from over 100 Hydrilla tubers collected at Cercle Nautique, Lake Tanganyika.
Limited rearing of other Hydrocharitaceae species was conducted. In Uganda, several species of Chironomidae were reared that were also produced by Hydrilla, including a single specimen of P. wittei (Table 3). In Burundi, several aquatic macrophytes have been found growing in association with Hydrilla, but not other members of the Hydrocharitaceae.
Voucher specimens have been prepared by both Epler and Copeland and distributed to our laboratories in Jinja, Uganda and Bujumbura, Burundi. The vouchers provide identified adult specimens of most Chironomid species (and all the common ones) and enable rapid identification of reared material by our non-specialist colleagues.


Table 1. Chironomidae reared from Hydrilla verticillata in Burundi




Subfamily

Tribe

Species




Chironominae

Chironomini

Kifferulus chloronotus (Kieffer)










Chironomus lindneri (Freeman)










Chironomus seydeli Goetghebuer










Dicrotendipes fusconotatus (Kieffer)










Dicrotendipes sudanicus (Freeman)










Kifferulus brevipalpis (Kieffer)










Parachironomus acutus (Goetghebuer)










Parachironomus dewulfianus (Goetghebuer)










Polypedilum cf. tenuitarsis










Polypedilum dewulfi Goetghebuer










Polypedilum wittei Freeman










Zavreliella marmorata (Wulp)







Tanytarsini

Cladotanytarsus pseudomancus










Rheotanytarsus guineensis Kieffer










Tanytarsus balteatus Freeman
















Orthocladiinae




Cricotopus cf. harrisoni Freeman
















Tanypodinae

 

Ablabesmyia cf. melaleuca Goetghebuer














Table 2. Chironomidae reared from Hydrilla verticillata in Ugandan lakes




Subfamily

Tribe

Species

Lake1

Chironominae

Chironomini

Chironomus callichirus Kieffer

Mut







Chironomus formosipennis Kieffer

Bis, Bun







Dicrotendipes chambiensis (Goetghebuer)

Bun







Dicrotendipes fusconotatus (Kieffer)

Bis







Dicrotendipes kribiicola (Kieffer)

Bun







Dicrotendipes peringueyanus (Kieffer)

Bun, Mut







Dicrotendipes septemmaculatus (Becker)

Bis, Bun







Harnischia cf. lacteiforceps Kieffer

Bis







Kiefferulus brevipalpis (Kieffer)

Bis, Bun, Mut







Kiefferulus chloronotus (Kieffer)

Bun







Kiefferulus fractilobus (Kieffer)

Bis, Bun, Mut







Nilothauma cf. latocaudatum Adam & Saether

Bis







Parachironomus acutus (Goetghebuer)

Bun







Parachironomus dewulfianus (Goetghebuer)

Bun







Polypedilum dewulfi Goetghebuer

Bis







Polypedilum micra Freeman

Bun







Polypedilum nr. tesfayi Harrison

Bun







Polypedilum sp.

Mut







Polypedilum vittatum Freeman

Bun







Polypedilum wittei Freeman

Bis, Bun







Xenochironomus trisetosus (Kieffer)

Bis







Zavreliella marmorata (Wulp)

Bis




Tanytarsini

Cladotanytarsus pseudomancus (Goetghebuer)

Bis, Bun, Mut







Rheotanytarsus ceratophylli Dejoux

Bis, Bun







Tanytarsus balteatus Freeman

Bis, Bun







Tanytarsus bifurcus Freeman

Bis







Tanytarsus flexistylus Freeman

Bis







Tanytarsus formosanus Kieffer

Bis, Bun







Tanytarsus harei Ekrem

Bis







Virgatanytarsus nigricornis (Goetghebuer)

Bun













Orthocladiinae




Cricotopus scottae Freeman

Bun







Nanocladius cf. saetheri Harrison

Bis













Tanypodinae




Ablabesmyia cf. dusoleili Goetghebuer

Bun







Ablabesmyia nilotica (Kieffer)

Bun

 

 

Ablabesmyia rimae Harrison

Bis

1Bis=Bisina, Bun=Bunyonyi, Mut=Mutanda






Table 3. Chironomidae reared from other Hydrocharitaceae species in Uganda Lakes

Lake

Plant species

Chironomidae species

Victoria

Egeria densa Planch.

Ablabesmyia nilotica (Kieffer)







Chironomini sp.







Chironomus callichirus Kieffer







Chironomus formosipennis Kieffer







Dicrotendipes fusconotatus (Kieffer)







Dicrotendipes kribiicola (Kieffer)







Dicrotendipes septemmaculatus (Becker)







Dicrotendipes sp.







Kiefferulus brevipalpis (Kieffer)







Kiefferulus fractilobus (Kieffer)







Rheotanytarsus ceratophylli Dejoux







Tanytarsus formosanus Kieffer










Bunyonyi

Lagarosiphon cf. ilicifolius Oberm.

?Tanytarsini sp.







Ablabesmyia cf. nilotica (Kieffer)







Chironomini sp.







Cladotanytarsus U-1







D. septemmaculatus







Dicrotendipes peringueyanus (Kieffer)







K. brevipalpis







Polypedilum wittei Freeman

 

 

T. formosanus


Lake Bisina surveys: Lake Bisina is a shallow water body with depth ranging from 0.6 to 4.3 m (average 2.6 m) and average clarity (Secchi reading) of 1.6 m. Water quality parameters are shown in table 4.
Table 4. Water quality parameters of Lake Bisina (means of 25 locations).


Parameter

Mean

Stdev

DO (mg/L)

3.8045333

0.9494847

Temp ( C )

27.056

0.8522693

pH

7.7132

0.3394733

Cond. (uScm-1)

242.89467

22.719639

TDS (mg/L)

121.62133

11.422472

Redox (mV)

-90.0067

19.488109

Nitrates (µg/L)

1252

259

Phosphates µg/L)

14.4

6.2

Ten plant species were found in surveys, with 9 submersed macrophytes and Nymphea nuchalli, a rooted plant with floating leaves. Najas horrida was the most widespread (97/112 locations) and abundant plant. Hydrilla was found at about one-half of sampled sites (57/112), and was the fourth most abundant plant (Figure 3). Two of the submersed plants were members of the Hydrocharitaceae; Najas horrida (formerly placed in Najaceae) and Ottelia scabra. Two plants have not yet been identified.




Figure 3. Average abundance of aquatic plants collected from 112 sites in Lake Bisina.
The distribution of Hydrilla was associated with water depth (Figure 4) with most Hydrilla found in shallower waters (mean depth 2.3 m), and never deeper than 3.4 meters. Hydrilla was always found in association with other plants (mean plants at sites with Hydrilla = 3.9) and was typically not the most abundant plant at locations where it was found.

Figure 4. Interpolation (inverse distance weighted) of water depth (left) and Hydrilla density (right) in Lake Bisina, Uganda.


Figure 5 Putative fish damage to hydrilla leaves.


The vast majority of insects reared from the samples are chironomids and to a lesser extent trichopterans, but these have not yet been identified. Dissections of 161 tips during the May survey revealed little or no obvious insect damage, but extensive feeding damage which could have been caused by foraging fish (Figure 4). Five greenish/red chironomid larvae were found on tips, although not associated with obvious damage. Tentatively, these larvae were identified as Dicrotendipes sp. Additionally, five immature trichopterans were found on plants.


Genetic studies
Microsatellite loci: We sequenced a total of 250 clones. One-hundred of these clones contained a microsatellite region. We then designed primers for a total of 35 clones that 1) had sufficient flanking regions around the microsatellite repeat to design primers, and 2) contained 10 or more consecutive microsatellite repeats since previous studies on a number of species have suggested at least this number of repeats is needed for the locus to be polymorphic. The majority of these clones (88%) were dinucleotide AGn repeat microsatellites. We then tested these markers with 4 - 8 Hydrilla individuals and visualized the products on 6% acrylamide gels stained with Gel Red (Biotium). Of these 35 primer sets, five did not amplify a product, 13 amplified a number of non-specific products or produced a smear, while 17 produced clear polymorphic banding patterns. We then fluorescently labeled one of the primers for each of these 17 loci to further screen their performance on the ABI 3130 Genetic Analyzer. Presently six of these loci have been tested on the Genetic Analyzer and four of these appear to give easily interpretable banding patterns. The other 11 loci are slated to be screened this month. The preliminary screening of Hydrilla samples using the four loci indicates that Florida Hydrilla and Hydrilla collected in the SE USA are triploid since these samples consistently give three DNA fragments at all these loci. The East African samples, on the other hand, appear to be diploid since they only give one or two fragments for each locus.
Screening for fluridone resistance: We successfully amplified a portion of the pds gene using nuclear DNA as judged by its perfect sequence alignment to the published cDNA sequence of the pds gene. All samples from Florida tested so far (N = 20) have the “wild” type or fluridone susceptible genotypes. Mike Netherland (USACE ERDC, Env. Laboratory) has recently sent us three fluridone resistant strains of Hydrilla for testing. Contrary to our expectations, we only found the non-mutated (fluridone susceptible) sequence in all of these samples even after multiple extractions and amplification attempts. Mike is currently retesting these strains for resistance and we are also extracting RNA from these samples and reverse transcribing it into DNA (cDNA) to capture the fully expressed pds gene to double check these results.

Discussion and future work
Surface guild insects have been rarely encountered in either country. This is not surprising considering that Hydrilla rarely reaches the water surface in the lakes of Uganda and Burundi. Therefore, we have concentrated our efforts on identifying sites that yield large enough numbers of target Polypedilum species to attempt colonization. In general, we have not reared the large numbers of Polypedilum that we expected based on the reports of Pemberton (1980) and Markham (1985). However, recently we have reared substantial numbers of Polypedilum from Hydrilla collected from Lake Tanganyika in November, 2007 and February, 2008. Repeat collections of this size, or larger, will provide enough Polypedilum to initiate colonization attempts in aquaria and cages that we have ready in both countries. Interestingly, both collections were made during periods when the lake level was low relative to that of other collections that we made. During these periods, Hydrilla was much closer to the water surface and, in one of our sites (Cercle Nautique), specimens sometimes reached the surface. During drier periods with lowered lake levels, it is possible that larvae that hatch from eggs deposited on the water surface by Polypedilum females have a greater chance of successfully locating Hydrilla. We plan to examine lake level records from the periods when Pemberton and Markham made Hydrilla collections to see if their rearing of large numbers of Polypedilum was made under similar conditions. If so, this would have positive implications for the possible control of Hydrilla by Polypedilum species in Florida, where this plant is usually found at or near the surface.
Except for the single example of leaf margin feeding associated with a pyralid larva, clear evidence of insect herbivory on Hydrilla has not been seen. In Burundi, Dicrotendipes fusconotatus was by far the most common species reared from Hydrilla. During examination and dissection of Hydrilla stems and growing tips during this reporting period and previously, larvae of D. fusconotatus are commonly found in poorly-defined feeding tubes associated with leaf whorls. Detritus and fecal material near the tubes can sometimes be mistaken for evidence of plant damage. However, it is very unlikely that this species is a herbivore (J. Epler, pers. comm.). We believe that the lack of finding actively feeding larvae in tips may be a function of the relatively small numbers of Polypedilum we have seen associated with Hydrilla. Insect herbivores are either very rare of highly seasonal. Although we will continue to examine Hydrilla growing tips for evidence of damage, we intend to concentrate our efforts on increasing the quantity of Hydrilla collected with the aim of rearing enough Polypedilum to establish a colony.
The plant diversity study in Lake Bisina demonstrated that Hydrilla grows in mixed plant communities with several other submersed aquatic plants, and its abundance is typically low to moderate. The distribution was correlated with water depth, with Hydrilla tending to occur in shallower areas of the lake, and never in the deepest areas. The water chemistry data collected from Lake Bisina needs to be compared to typical values in Florida lakes infested with Hydrilla, as this may suggest abiotic factors which influence density.
Herbivory by fish seems to be a strong possibility, as many leaves collected in both Uganda and Burundi appear to have been chewed. Reports from the two previous Hydrilla natural enemy surveys in East Africa (Pemberton and Markham) also mentioned the possibility of fish herbivory. Fisheries experts at the National Fisheries Resource Research Institute, where the project is based in Uganda, believe that some species of small haplochromine fish (sub-family of Cichlidae) may feed on Hydrilla. We intend to test this hypothesis by trapping fish using gill nets in areas of Lake Bisina where Hydrilla grows and dissecting gut contents. If Hydrilla is found in fish stomachs, an attempt will be made to colonize these fish in aquaria with Hydrilla and other plants for feeding studies.
The genetic studies are progressing well. We expect to have approximately 10 useable microsatellite loci after testing the final 11 loci. We will then genotype all samples at these loci and analyze the population genetic structure of USA and African samples. We also need to finish screening new African samples (N = 30), SE Asian and Australian samples (N = 40) and some USA samples (N = 20) at the chloroplast regions that were described in the 2006 DEP report. The population genetic data from the nuclear DNA microsatellites and chloroplast DNA will then be compared for the entire dataset. The previous results for the cpDNA data indicated that the African, Floridian, and Indian samples were identical. Our preliminary microsatellite work however indicates that there may be a ploidy difference in the nuclear DNA between the African and Floridian samples. The Florida Hydrilla results are consistent with the findings of a recent study by Puri et al. (2007b) that used flow cytometry to estimate ploidy levels in Florida Hydrilla and determined that it was most likely to be triploid.
One possible explanation for our unexpected sequencing results for the pds gene in fluridone resistant strains is that since Florida samples are triploid, there may only be one copy of the mutated gene that confers resistance and two copies of the non-mutated gene. The two non-mutated copies may then be more likely to amplify during PCR (because of their greater numbers in the sample). Another possibility is that there are a number of nonfunctional copies of the pds gene (“pseudo-genes”) in the nuclear genome which would also be more likely to be amplified during PCR. If our sequencing of the cDNA pds gene in fact reveals the mutation, then we will attempt to optimize the PCR conditions to target the copy of the pds gene that contain the mutation. We then hope to develop SNP (single nucleotide polymorphism) markers that are specific for the different described mutations in the pds gene that confer resistance to fluridone. Once developed, these markers are relatively inexpensive and easy to use, and a large number of samples could potentially be screened quickly for the resistant biotypes of Hydrilla.
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