Aquatic Ecology 34: 413–420, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

413

The use of Solid Phase MicroExtraction (SPME) devices in analysis for

potential mosquito oviposition attractant chemicals from cyanobacterial
mats

Eliska Rejmankova1, Richard M. Higashi2, Donald R. Roberts3 , Michele Lege1 and

Richard G. Andre3
1 Department of Environmental Science and Policy, University of California, Davis, CA 95616, USA (E-mail:
erejmankova@ucdavis.edu);
2 Environmental Chemistry Group, Crocker Nuclear Laboratory, University of California, Davis, CA 95616, USA;
3 Department of Preventive Medicine and Biometrics, USUHS, 4301 Jones Bridge Rd., Bethesda, MD 20814, USA

Accepted 1 December 2000

Key words: Anopheles albimanus, cyanobacterial mats, aliphatic alcohol, oviposition attractant, SPME device

Abstract
Females of Anopheles albimanus mosquito are known to prefer floating cyanobacterial mats over open water for
oviposition. We used Solid Phase MicroExtraction (SPME) devices on-site to trap substances volatilized from
these two environments, followed by analysis by GC-MS (gas chromatograph-mass spectrometer). In enclosed
headspace field microcosms, an unidentified C-15 aliphatic alcohol was persistently found over cyanobacterial
mats as compared to open water. Based on this finding, we conducted oviposition experiments using a commercially
available compound, n-pentadecanol, close in molecular weight and mass spectral pattern to the unknown C-15
aliphatic alcohol. The results indicate a tendency of female mosquito to oviposit more eggs in containers with the
tested chemical as compared to water and blank.

Introduction northern Belize that showed a strong preference of a

Cyanobacterial mats (Cyanophytes, blue-green algae)
are an important component of extensive marshes in
the Yucatan Penninsula (Rejmánková et al., 1996a),
specifically those dominated by sparse emergent
macrophytes (Eleocharis spp.). The mats consist of
fine filaments of cyanobacteria from the genus Lep-
tolyngbya which form most of the biomass and are
intermingled with many other species of cyanobacte-
ria, diatoms, and bacteria. Cyanobacterial mat com-
munities are generally characterized by high metabolic
rates including photosynthesis during the day and res-
piration during the night. In addition, other activities
such as nitrogen fixation and sulfate reduction are
common in these mats (Cohen & Rosenberg, 1989;
Rejmánková & Komárková, 2000). In our earlier pa-
per (Rejmánková et al., 1996b) we reported results of
a field oviposition experiment conducted in a marsh in
malaria-transmitting mosquito, Anopheles albimanus,
for floating mats of cyanobacteria.
While we confirmed that the reason for the fre-
quent presence of A. albimanus larvae in cyanobacter-
ial mats was the strong tendency of mosquito females
to oviposit there, the question remained why fe-
males choose cyanobacterial mats for oviposition. The
oviposition behavior of mosquitoes has been widely
studied and results indicate that many species are
capable of using tactile, chemotactile, olfactory, or vi-
sual cues to assess such site characteristics as color,
reflectance, texture, moisture, salinity, surrounding
vegetation, bacterial growth, conspecific population
density and the presence of a wide variety of chem-
icals (Bentley & Day, 1989; Beehler et al., 1993;
Dhileepan, 1997). Attention of many researchers has
focused on testing various organic infusions (hay, alfa-
alfa, bermuda grass) that have been shown to attract
414

ovipositing females (Millar et al., 1992; Lampman &

Novak, 1996).
The information on some oviposition attractants
is available for several species of genus Culex and
Aedes, but no chemical oviposition attractants have yet
been identified for anopheline mosquitoes. Dhar et al.
(1996) report that volatile substances from natural
products, such as neem, reetha and garlic, suppress
oviposition of Anopheles stephensi and A. culcifacies.
Only a few anopheline oviposition experiments have
been conducted in the field (Orr & Resh, 1992).
According to Bentley and Day (1989), there are
many similarities between mosquito host-seeking and
ovipositing behaviors. Mosquitoes make use of CO2
odor plumes to locate hosts. We ruled out the possibil-
ity of CO2 being the oviposition attractant because re-
peated measurements showed that the differences be-
tween CO2 emitted from cyanobacterial mats vs. open
water were not statistically significant (Rejmánková,
unpublished data).
Our objective was to find whether cyanobacterial
mats release specific substances that are not present
Figure 1. Diagram of a sampling chamber, showing the fine detail
in the non-mat environment and that could potentially of SPME device.
serve as oviposition attractants. To accomplish this
we used an operationally simple device for collecting
volatile organic compounds on a sorbent trap, Solid cyanobacterial mats. It further provides data on ovipo-
Phase MicroExtraction device (SPME), followed by sition experiments using compounds similar to our
direct desorption of chemicals trapped on the de- as-yet unidentified aliphatic alcohol.
vice into a gas chromatograph for analysis (Millar
& Haynes, 1998). This trapping device is readily
deployed at remote sites since it is compact and Material and methods
self-contained, and the method with one-step sample
trapping and no further sample preparation minimizes The study was conducted in several marshes on the
losses and artifacts from sample storage and/or ex- Northern Coastal Plain of Belize. Detailed descrip-
traction of volatile components. A similar method has tion of the area is given in Rejmánková et al. (1993,
been previously used to analyze the odor components 1996a). Three marshes, Chan Chen (CH), Buena Vista
of flowers of Arum maculatum (Kite, 1995), semio- (BV) and Doubloon (DBL) with extensive cyanobac-
chemicals released by Phyllonorycter sylvella moths terial mats were selected as study sites. We have many
(Borg-Karlson & Mozuraitis, 1996) and Lepidoptera records about Anopheles albimanus larvae presence in
(Frerot et al., 1997). these marshes (Rejmánková et al., 1996b).
To test possible behavioral effects of compounds SPME devices of 100 µm polymethylsiloxane
related to those released from cyanobacterial mats phase (Supelco Inc., Bellefonte, PA, USA), which re-
we conducted oviposition experiments using wild fe- semble and are used like syringes, were exposed in
males. We choose wild specimens rather than a col- closed round PVC chambers (25 cm diameter ∼ 25 cm
onized population whose behavior is often modified above water; the headspace volume calculated as a
by various factors, such as reduced heterozygosity volume of a cylinder) equipped with flotation collars
(Chareonviriyaphap et al., 1997). and floating either in algal mats or in open water (Fig-
This paper reports on the utility of the SPME ure 1). The devices were inserted in each chamber
volatile chemical trapping technique in remote loca- through a septum and left exposed there for 4 h af-
tions under adverse in-situ conditions, and the results ter sunset (18:30–22:30), during the time of reported
obtained from measuring volatile compounds from oviposition for Anopheles albimanus (Chadee et al.,

1993). Devices for field blanks were handled similarly,
except that they were not exposed in the chambers. In
each marsh we used two chambers over algal mats and
one over open water. The measurements were con-
ducted in the BV marsh in October 1995 and in all
three marshes in February 1996 and August 1996.
After exposure the devices were capped with a sep-
tum (MicroSep F174N, Alltech Inc., Deerfield, IL,
USA). The entire assembly was stored in airtight poly-
carbonate centrifuge vials (Fisher Scientific, Pittsburg,
PA, USA) and transported on ice to UC Davis. The
time interval between the exposure and processing
at UC Davis was less than a week. With no further
processing, the SPME devices were inserted into the
gas chromatograph (GC) injector for thermal desorp-
tion as prescribed (Martos & Pawliszyn, 1997; Steffen
& Pawliszyn, 1996). Analysis used a Varian 3400 GC
interfaced to a Finnegan ITD 806 ion-trap mass spec-
trometer with electron-ionization (EI-MS) at 70 eV,
scan from 46–400 mass units, four full scan spec-
tra/sec averaged into one spectrum/sec, and automatic
gain control set to 45 amu. Column was 0.15 mm
× 50 m dimension, ‘DB-5 equivalent’ (5% phenyl-
methyl silphenylene siloxane copolymer) coat that
was 0.4 microns thick (BPX-5 column, SGE, Inc.,
Austin, TX, USA). Temperature program was 40 ◦ C
with 4 min hold, then 10 ◦ C/min rise until 300 ◦ C.
Hydrogen at 40 cm/sec velocity was used as a carrier
gas, injector was 280 ◦ C, and the transfer line was
300 ◦ C.
Peaks from GC/EI-MS runs were assigned tenta-
tive identities based on mass spectral similarity scores
with spectra from a 64,000 entry NIH/EPA/NIST mass
spectral library, using the search algorithm of the in-
strument software (Trapmaster, Finnegan MAT Inc.,
San Jose, CA, USA).
To qualitatively verify the results using SPME de-
vices in the field, corresponding samples of mats
and open water for the February 1996 sampling were
sealed on-site in airtight Teflon-lined silicone septum-
capped borosilicate glass vials. The headspace was
then analyzed using the same SPME device method.
As with the field trapping of volatile constituents, the
SPME devices were inserted through the septum, ex-
posed for 15 min, and analyzed as described above.
GC/chemical ionization MS (GC/CI-MS) was addi-
tionally employed to obtain further information on the
vial samples. CI-MS detection has a far higher prob-
ability than EI-MS of detecting the molecular ion and
initial loss fragments. For the classes of volatile com-
pounds detected over the algal mats, this translates
415
into greater confidence regarding the molecular weight
and further evidence of the chemical functionality or
class. The GC parameters for this procedure were as
listed above, and the MS parameters were: manifold
= 220 ◦ C, electron energy = 70 eV, electron emission
current = 10 microA, isobutane reagent gas delivery
pressure at 5 psi, maximum reagent gas ionization
time = 3000 msec, ionization level = 8.6 amu, ratio
of reagent ions 57m/z:43m/z was set to 0.4, maximum
reaction time = 250 msec, reaction level = 23 amu,
ion rejection mass = 99 amu, with automatic reac-
tion control full scan acquisition from 99–650 m/z at a
rate of two spectra/sec, which were averaged into one
spectrum/sec.
Analysis of on-site headspace traps has many un-
certainties and although the data pointed to a pen-
tadecanol, we were not able to determine which
of the many possible isomers of pentadecanol was
present. We decided to conduct an experiment with n-
pentadecanol, the only readily available pentadecanol
isomer, although we knew from GC-MS analysis that
the chemical identified by SPME trapping in the field
was a different isomer than n-pentadecanol. Ethanol
was selected as the co-solvent to aid in dispersion
of n-pentadecanol in water, as ethanol occurred in
the headspace at all sites (Figure 3). At field sites,
0.24 ml of ethanol stock solutions of n-pentadecanol
were dissolved in 240 ml of water to provide the
final concentration corresponding to an estimate of
the source concentration of the unknown compound
(assuming that the source is water), which worked
out to approximately 44 microM. This estimate was
based on approximate mass of the C-15 aliphatic alco-
hol trapped on the SPME device (10–40 pg, assum-
ing similar MS response as that of n-pentadecanol)
at 100% recovery, and given the trapping chamber
volume, back-calculating the potential water concen-
tration (Thomas, 1982) using Henry’s Law coefficient
for n-pentadecanol (estimated using Molecular Analy-
sis Pro software, NorGwyn Montgomery Software,
North Wales, PA, USA). The test solutions of chem-
ical, ethanol blank, and water control, each with three
replicates, were placed in white 250 ml Teflon beakers
randomly positioned in the oviposition cage.
Anopheles albimanus females were collected with
hand-held aspirators from investigators’ legs. They
were provided with two bloodmeals (in 24-h intervals)
and sugar solution ad libitum. The mosquitoes were
kept either in a cage covered with dark cloths outdoors
or in the unlit room in our field laboratory in Orange
Walk, Belize. Forty eight hours after the second blood-
416
meal, the blood-engorged females were released into
the oviposition cage (60×60×60 cm) after sunset. The
eggs were counted the first, second and third morn-
ings following the release. In one case we counted the
eggs the first and fourth mornings. Dates of individ-
ual oviposition experiments and numbers of females
used are presented in Table 1. Differences of means
between egg counts were evaluated with Scheffe’s test.
Results
Survey of SPME device and vial headspace contents
from the field
Figure 2 shows a typical chromatogram of SPME
analysis of a chamber headspace, illustrating an un-
crowded chromatogram; almost all components were
unambiguously baseline separated. Figure 3 shows re-
sults from the February 1996 sampling, which is the
most reliable because each component was verified to
be associated with field samples, as described below.
The SPME device analyses of headspace over mats
and water revealed at least 14 components not found
in field blanks, plus the synthetic insect repellent N,N-
diethyltoluamide (‘DEET’), (Figure 3). Among these,
there was only one component that was always higher
in headspace over cyanobacterial mats than in those
over water without mats (Figure 3), a trend that was
supported by the other samplings (data not shown).
All other components over mats varied considerably
relative to over water (e.g. Figure 3).
Headspace analyses from larval habitat material,
sealed on-site in Teflon-lined septum vials, was per-
formed to qualitatively verify the association of these
peaks with field samples, and to obtain higher con-
centrations for verifying and improving mass spectral
data. All compounds detected by SPME device field-
trapping were present in samples from their respective
sites, and therefore were not artifacts of the procedure.
Furthermore, these samples provided the opportunity
for re-analysis under different GC-MS conditions to
obtain additional chemical information. We chose to
reanalyze using GC/CI-MS. For the compound em-
phasized in Figure 2, the GC/EI-MS analysis con-
tributed information that this volatile compound had
an aliphatic portion of >11 carbons, probably not
branched heavily. The GC/CI-MS analysis yielded
complementary information which indicated a mole-
cular weight of 228 and the likelihood of an -OH
functional group (indicated by a prominent loss of 18
mass units, or water). The evidence did not support
structural alternatives that are common volatile classes
from biota, such as ketones, aldehydes, esters, iso-
prenes, or sterols. This information fit a pentadecanol
compound as a prime candidate. However, the GC re-
tention time of the peak unique to algal mats did not
match with authentic n-pentadecanol standard, so it is
probably one of the other isomers of pentadecanol.
Oviposition experiments
The oviposition experiments ran into many difficul-
ties, the chief of which was high mortality of captive
mosquito, which caused several planned tests to be
aborted. Table 1 reports results from the three repli-
cated experiments showing the final numbers of eggs
deposited into experimental containers the second and
in one case the fourth morning after releasing fe-
males into the oviposition cage. The variability in the
numbers of eggs oviposited was large and individual
experiments treated separately did not show significant
differences due to a small number of replicates except
for the second experiment (Table 1). When the egg
counts were standardized by the number of females re-
leased into the oviposition cage and total means from
all three experiments calculated, the n-pentadecanol
treatment was significantly different from both water
and ethanol blank (Figure 4).
Discussion
Field survey of substances volatilized from
cyanobacterial mats
This is the first demonstration of collecting volatile
compounds from mosquito larval habitats on SPME
devices followed by direct gas chromatographic analy-
sis. Conventional trapping methods require consid-
erably more equipment and utilize extraction tech-
niques that can impart artifacts resulting from the
extraction/distillation procedures (Kite, 1995). Our
main objective stated in the introduction, i.e., to find
whether the air over cyanobacterial mats contains spe-
cific substances that are not present or in very low
concentration over non-mat environment, has been
accomplished. The technique proved to be feasible
and relatively easy to implement for in situ qualita-
tive assessment of substances volatilized from larval
habitats. The compound that was characteristic for
the cyanobacterial mat environment was tentatively
assigned as a C-15 aliphatic alcohol. We were not
 417

Figure 2. GC/EI-MS total ion current tracing of SPME device analyses, from mat and water headspaces of the DBL site. Analysis conditions
are stated in the Methods section. The inset shows the mass spectrum of an unknown C-15 aliphatic alcohol.

Figure 3. Abundance of components in the headspace over mats relative to over water, as detected by GC/EI-MS analysis of SPME devices,
in duplicate analyses of the three sites in February, 1996. The presentation order of components is arbitrarily based on increasing GC retention
time. Most of the components are of unverified identity except for the 1st (ethanol) and 10th (the insect repellent DEET) from the left. The solid
black bar (12th component, designated by stars) is the C-15 aliphatic alcohol. CH = Chan Chen, DBL = Doubloon, BV = Buena Vista.
418
Table 1. Results of three oviposition experiments showing the number
of eggs found in treatment containers on the second morning (March
1997; July 1997) and fourth morning (August 1997) after releasing fe-
males into the oviposition cage. Number of females per cage is indicated
in parentheses. Differences of means between egg counts were evaluated
with Scheffe’s test. Means sharing the same letter are not statistically
different (P > 0.05)

Date Replicate N-Pentadecanol Blank Water

March 1997 1 42 0 40
(15) 2 2 0 0
3 14 0 0
Mean 19.3 a 0a 13.3 a

July 1997 1 172 202 17

(50) 2 401 8 1
3 246 10 5
Mean 273.0 a 73.3 ab 7.7 b

August 1997 1 46 14 14
(20) 2 87 22 50
3 20 10 14
Mean 51.0 a 15.3 a 26.0 a

able to identify the compound more precisely; this
would require substantial investigation involving col-
lections of larger amounts of headspace in conjunction
with other analytical techniques, potentially custom
chemical synthesis, and numerous bioassays.
Factors affecting female choice of ovipositing site
may include availability of food for rapid larval de-
velopment (Blaustein & Kotler, 1993). Bacteria have
been considered as the most important component of
food of mosquito larvae (Merritt et al., 1992) so it is
not surprising that females would choose a site rich in
bacterial fauna such as cyanobacterial mats. The com-
pound tentatively assigned here is neither an indole
(Millar et al., 1992) nor a aliphatic lactone (Laurence
& Pickett, 1982), the two chemical classes associ-
ated with oviposition behavior. For volatile mixtures,
mosquito species (e.g. Culex pipiens) responded to
products released from protein degradates (Beehler
et al., 1994) and by bacterial cultures of Pseudomonas
(Rockett, 1987). However, as compounds were not
identified, this does not rule out the possible presence
of long-chain alcohols in those studies.
Oviposition experiment
Majority of oviposition experiments reported in the
literature have been conducted in the laboratory. As
Bentley & Day (1989) emphasize, these studies do
not necessarily reflect mosquito behavior in the field
and many described behaviors may be laboratory ar-
tifacts without relevance to natural situations. In our
oviposition experiments, we choose a compromise:
we used wild females who were left to oviposit in
an oviposition cage placed either outdoors or indoors.
We endured a high rate of failure caused by various
factors such as high female mortality, or oviposition
bias due to a slightly different amount of light in
one part of the cage. What have we learned from
these experiments? It seems probable that the class of
chemicals that we identified and used for experiments
has a potential for providing short-distance stimuli for
ovipositing females. Several factors could have been
responsible for not very clear-cut results: 1) The test
compound we used was of the same chemical class,
but known to be different from the one detected in the
SPME device traps. 2) The concentrations used were
only gross estimates involving multiple assumptions,
and could have been very different from the natural
situation. Knight & Corbet (1991) provided evidence
that with increasing concentration of hexanoic acids
tested on Aedes aegypti there is a shift from attractancy
to repellency. Unfortunately, they did not determine
the concentrations in the air. 3) There may be more
than one oviposition cue and while the chemical stim-
uli were provided, the other cues were not. McCrae

Figure 4. Number of eggs deposited into experimental containers.
Plotted values are means from the three experiments (see Table 1
for primary data). Error bars indicate SE. Columns sharing the same
letter are not significantly different from each other (P > 0.05;
Scheffé’s test).
(1984) points out that some stimuli may elicit response
only in a cumulative fashion. Thus, the artificial ovipo-
sition environment may have caused females to behave
atypically.
Concentration of volatilized substances as oviposition
cues
Mosquito reproductive behavior is a complex process
including finding a host for blood meal and subsequent
selection of an aquatic place for egg-laying (Klow-
den, 1990). The exogenous stimuli that initiate these
behaviors include host odor as well as visual and
chemical (volatile components) cues characteristic of
oviposition sites. The females have to be first attracted
to a potential oviposition site from a distance (pre-
oviposition). This long-range attraction is probably
mediated mainly by visual cues (Isoe et al., 1995),
while the actual oviposition site selection may in-
volve short-range chemical and/or visual stimuli. The
type of experiments we conducted cannot determine
how mosquito females find the oviposition site from a
distance.
Working on the hypothesis that cyanobacterial
mats (but not any loosely floating low concentra-
tion cyanobacteria in open water) provide close-range
stimuli for oviposition of Anopheles albimanus, our
previous oviposition experiment conducted in the field
showed that females were positively discriminating
cyanobacterial mats vs. open water (Rejmankova et al.,
1996a). The floating mats are often quite patchy rang-
ing in size from about 10 cm diameter and up: the
patchiness would require a female to get close for
discrimination. The estimated concentration of the C-
419
15 aliphatic alcohol in the headspace, based on what
was absorbed by SPME devices, was low, calculated
to be <40 pg from the 12.26 L of headspace, or <
743×10−12 M. Unfortunately, it is difficult to compare
this figure with previous studies, as concentrations in
air were rarely, if ever, determined.
However, the results of the on-site bioassays,
which used 44 microM in water based on the
headspace analysis, can be compared with previous
studies which used similar experimental designs. The
concentration we used is up to three orders of mag-
nitude lower than is often used for attractancy and
repellency experiments in the field and laboratory
(cf., Knight & Corbet, 1991), but one to six orders
of magnitude higher than reported for Culex attrac-
tance using 3-methylindole (Millar et al., 1992). It is
possible that in natural settings compounds such as
3-methylindole are relatively distant attractants, and
the other reported classes of compounds including the
C-15 aliphatic alcohol tentatively identified here, are
close-range stimuli for the corresponding species and
habitats.
In summary, this article introduces the option of
using the SPME devices to trap substances volatilized
from larval mosquito habitats. Although a vital first
step, it is part of a long process of screening the
volatile chemicals, identifying their chemical nature,
conducting oviposition tests, and, eventually, using the
identified attractants in oviposition traps for the actual
vector control.
Acknowledgements
Funding for this project was provided, in part, by
the University of California Research Expedition Pro-
gram (UREP), U.C. Davis Faculty Research Grant,
the Uniformed Services University of the Health Sci-
ences through grant R087DB, and UC-Davis Cen-
ter for Ecological Health Research (USEPA grant
#R819658). We thank John Grieco, Nicole Achee, and
Daniel Rejmánek for help with collecting mosquitoes.
Comments of three anonymous reviewers helped to
improve the manuscript.
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