Elsevier

Acta Tropica

Volume 115, Issue 3, September 2010, Pages 227-233
Acta Tropica

Species identification of plant tissues from the gut of An. sergentii by DNA analysis

https://doi.org/10.1016/j.actatropica.2010.04.002Get rights and content

Abstract

There are three commonly used assays to identify plant material in insect guts: the cold anthrone test for fructose, the cellulose staining test for visualizing plant tissue and gas chromatography for seeking unique sugar content profiles. Though sugar and cellulose tests can distinguish between the general sources of sugar meal (nectar versus tissue), they cannot identify the species of plant sources. Even gas chromatography profiles can be problematic; there are reported instances of intra-specific variation as well as inter-specific and intergeneric variation that can mar results. Here, we explore the potential for DNA analysis to help resolve this issue. First, Anopheles sergentii were exposed to branches of two species of highly attractive flowering bushes in the laboratory and the great majority (∼90–98%) were positive for sugar from nectar while very few were positive for cellulose (∼0.5–8%) and DNA (6–19%). Moreover, laboratory An. sergentii showed opposing preferences, tending to obtain sugar from nectar of one plant (Tamarix nilotica) but to feed more on tissue from the other (Ochradenus baccatus). An. sergentii are exposed to a wide variety of plants in their natural desert habitats and in the absence of flowers in the dry season, they resort to feeding specifically on tissues of a few plants. According to DNA analysis the favorite plants were Suaeda asphaltica, Malva nicaeensis and Conyza dioscoridis, which are succulents that account for less than 1% of vegetation in the area.

Graphical abstract

In this study, DNA analysis was used to determine the species of plant meals found in the guts of laboratory fed and field-caught Anopheles sergentii.

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Introduction

The energy for the activities of mosquitoes is provided by sugars (for a review see Foster, 1995) which are obtained primarily from floral nectar. Other sources are extra floral nectaries, honeydew excretions of homopterans or exudates from damaged fruits and leaves (McCrae et al., 1976, Mogi and Miyagi, 1989). Mosquitoes are even known to pierce plant tissues to obtain sugar-containing liquid (Abdel-Malek, 1964, Magnarelli, 1979).

Schlein and Jacobson (1994) and Schlein and Müller (1995) described a method for staining cellulose residues in the dissected guts of sand flies and mosquitoes. When combined with a method that measures gut sugar content, Müller and Schlein (2005) were further able to demonstrate variation between nectar feeding and plant tissue feeding according to season and habitat. Neither of these tests however, could identify the species of plant ingested.

DNA fingerprinting of plant meals is a promising, yet unexplored, method of identifying the components of the sugar diet of insects. The presence of plant material in the form of cellulose in insect guts is significant because it suggests that plant DNA may also be ingested during the meal. In previous studies, we tested this hypothesis and demonstrated that a short fragment of chloroplast DNA could be extracted and identified from the guts of various insects including crickets, grasshoppers, locusts, beetles, aphids, cicadas and caterpillars (Miller et al., 2006, Matheson et al., 2008).

During the dry season (summer and autumn) in arid areas of Israel, few perennial trees and bushes are in bloom. We assumed that in response to nectar scarcity, plant tissue meals become indispensable sugar sources for mosquitoes. This assumption was confirmed when it was found that during the summer and fall, 46.3% of Anopheles sergentii females caught in the lower Jordan Valley desert fed on plant tissue, while during the short but extensive blooming period after the winter rains, only 0.5% of An. sergentii females were plant tissue positive (Müller and Schlein, 2005).

Here, we use a molecular approach to identify the DNA of specific plant species in the gut of An. sergentii caught in the southern Jordan Valley desert in autumn. To this end, we screened laboratory fed and field-caught mosquitoes for a 157 bp fragment of the ribulose bisphosphate carboxylase gene large subunit (rbcL) and compared the resulting sequences to a reference collection of 24 common plant species from the southern Jordan Valley, whose unique rbcL sequence fragments were characterized in a previous study (Matheson et al., 2008).

Section snippets

Field site and mosquitoes

Male and female An. sergentii were collected in the autumn by UV lighted CDC traps (model 1012 John W. Hock Company, Gainesville, Florida) in the southern Jordan Valley, Israel, at an altitude of approximately −350 m below sea level (b.s.l.).

For laboratory experiments blood fed An. sergentii were trapped near Jericho in a tent in which goats were gathered for the night. The trapped mosquitoes were maintained under insectary conditions for oviposition (Beier et al., 1987). Before experiments,

Laboratory fed mosquitoes: T. nilotica versus O. baccatus

Flowers of T. nilotica and O. baccatus are very attractive to the local mosquito fauna in general and An. sergentii in particular (Müller and Schlein, 2006). To determine if mosquitoes preferred to feed on one of these plants over the other, we conducted the assays on mosquitoes exposed to either T. nilotica or O. baccatus flowering branches in the laboratory (Table 1).

There were significantly more mosquitoes feeding on nectar of T. nilotica than O. baccatus as measured by the cold anthrone

Discussion

Sugar feeding is integral to the longevity, fecundity, host seeking behavior and other activities of biting insects such as mosquitoes and sand flies (McCrae et al., 1976, Mogi and Miyagi, 1989, Foster, 1995, Schlein and Müller, 1995). Thus the sugar sources can be used for control and decrease of disease transmission by their removal (Foster, 2008), by spraying them with sugar and toxin (Schlein and Müller, 2008) or by using attractive sugar baits (Müller et al., 2010). However, the preferred

Acknowledgements

The authors wish to thank Professor Avinoam Danin for his plant photos. The Bill and Melinda Gates Foundation grant number 0304721 partially supported this project.

References (27)

  • P.R. Grimstad et al.

    Nectar sources of Wisconsin mosquitoes

    J. Med. Entomol.

    (1974)
  • T.A. Hall

    BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT

    Nucleic Acids Symp. Ser.

    (1999)
  • D.E. Impoinvil et al.

    Feeding and survival of the malaria vector Anopheles gambiae on plants growing in Kenya

    Med. Vet. Entomol.

    (2004)
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