Oviposition response of Lutzomyia (Lutzomyia) renei (Martins, Falcão & Silva) (Diptera: Psychodidae) to extracts of conspecific eggs in laboratory bioassays

Jeane Cristina Menezes Alves¹, James G C Hamilton², Reginaldo Peçanha Brazil³

Recibido: 24-ii-2003
Aceptado: 13-iii-2003
Correcciones devueltas por el autor: 28-vii-2003

Menezes A JC, Hamilton C JG, Peçanha B R. 2003. Oviposition response of Lutzomyia (Lutzomyia) renei (Martins, Falcão & Silva) (Diptera: Psychodidae) to extracts of conspecific eggs in laboratory bioassays. Entomotropica 18(2):121-126.

In this study bioassays were carried out to evaluate the possible attractive and/or stimulant effect of extracts of the eggs of gravid female Lutzomyia renei. The response to the oviposition pheromone by L. renei was not as marked as that found for L. longipalpis and Phlebotomus papatasi. Extracts equivalent to 100 or 200 eggs did not produce a perceptible attraction and/or stimulation to oviposit, although extracts of 1000 eggs did produce a slight attractive response. Chemical analysis of L. renei egg extracts revealed the presence of various fatty acids and complementary bioassay experiments are needed to prove a possible stimulant effect.

Additional key words: Biology, pheromone.

Resumo

Menezes A JC, Hamilton C JG, Peçanha B R. 2003. Resposta à oviposição de Lutzomyia (Lutzomyia) renei (Martins, Falcão & Silva) (Diptera: Psychodidae) aos extratos de ovos coespecificos em bioensaios de laboratório. Entomotropica 18(2):121-126.

Neste estudo foram realizados bioensaios para avaliar o possível efeito atrativo e/ou estimulante de extratos de ovos de Lutzomyia renei. A resposta ao feromônio de oviposição pela L. renei não foi detectada como para L. longipalpis and Phlebotomus papatasi. Extratos equivalentes a 100 ou 200 ovos não produziram atração perceptível e/ou qualquer estimulo à oviposição, embora extratos de 1000 ovos produziram um leve estimulo atrativo. Análise química dos extratos de ovos de L. renei revelaram a presença de vários ácidos graxos e experimentos complementario são necessários para provar o possível efeito estimulante.

Palavras chaves adicionais: Biologia, feromônio.

Introduction

The hematophagous Diptera are non-social insects that, with few exceptions, do not show protective maternal behavior. Selection of the oviposition site is therefore crucial to maximize survival of the progeny. It has been suggested that semiochemical attractants (apneumones and oviposition pheromones) are used by many insect species to help the female orientate towards appropriate oviposition sites (McCall & Cameron 1995).

Oviposition pheromones have been demonstrated in several species of Culex, (Starratt & Osgood 1972, 1973; Bruno & Laurence 1979; Laurence & Pickett 1982,1985) Simulium (Coupland 1992; McCall et al. 1994; McCall 1995), and Aedes (Allan & Kline 1998) as well as other hematophagous insects (Barton Browne et al. 1969).

Among phlebotomine sand flies however, the chemical identification of the oviposition pheromone has been restricted to a single species, Lutzomyia longipalpis (Lutz Neiva). The oviposition pheromone was demonstrated in several laboratory experiments where eggs, or hexane extracts of eggs, showed an attractant and/or stimulant effect on gravid conspecific females (Elnaiem & Ward 1990, 1991 Elnaiem et al. 1991). The same effect was also observed when extracts of gravid female accessory glands were used in a similar bioassay and suggested that these glands probably constituted the site of production of the pheromone and further suggested that the pheromone was deposited on the eggs at the moment of oviposition (Dougherty et al. 1992). Recently, the L. longipalpis oviposition pheromone was identified as dodecanoic acid, a simple low molecular weight, unbranched fatty acid (Dougherty & Hamilton 1997).

Lutzomyia renei (Martins, Falcão & Silva, 1957), found in Brazil, principally in Minas Gerais State, is an anthropophilic species closely related to L. longipalpis (Martins et al. 1978). Although L. longipalpis and L. renei belong to the subgenus Lutzomyia, L. renei is a member of the related series (renei), which does not possess secretary glands (Lane & de Souza Bernardes 1990). A recent study showed that L. renei males have a single pheromone disseminating structure (Spiegel et al. 2002) but do not produce terpenoid compounds used as sex pheromones in L. longipalpis (Hamilton et al. 1999). We have also observed that L. renei do not `wing flutter' behaviour also associated with sex pheromone production. Together these results would suggest that although L. longipalpis and L. renei are closely related their chemical ecology is significantly different.

The objective of the present study was to further investigate aspects of the chemical ecology L. renei. In particular we wished to determine if L. renei females produce an oviposition pheromone. Therefore we undertook a series of oviposition bioassays in an attempt to evaluate the attractive and/or oviposition-stimulating effect of extracts of conspecific eggs on gravid females of this species.

Materials and Methods

Sand flies: The sand flies used in the bioassays were collected from a calcareous rock fissure at Lapinha Caves (municipality of Lagoa Santa, Minas Gerais State, Brazil, 19º 37'S; 43º 53'W); using CDC light traps baited with quail (Coturnix japonica), between 16.00 - 09.00. Females collected overnight were blood fed on hamsters anesthetized with 0.7 ml thiopental and subsequently transferred to holding pots (8cm in height x 11 cm in diameter). A honey:water (1:1) mixture on cotton wool was provided as a sugar source and males and females were maintained in the holding pots in plastic boxes in an incubator (25-26ºC; 90% relative humidity (RH); photoperiod 12h:12h). Four days after taking a blood meal, gravid females were used in the experiments. After completion of bioassays females were dissected, under a dissecting microscope, in a drop of Berlese solution for examination of spermathecae and cibarium to confirm the L. renei identifications.

Preparation of egg extracts: Extracts for bioassays were made using 1-2 day old L. renei eggs. The eggs were laid by the females directly onto filter paper discs maintained inside holding pots and subsequently transferred in quantities of 100, 200 and 1000 to glass ampoules prepared from Pasteur pipettes. After introduction of the eggs, 100µl hexane (HPLC - EM Science/Germany) was added to the ampoules, which were then heat-sealed in a Bunsen flame and maintained at -20ºC until they could be used in the bioassays.

The extracts destined for use in bioassays to be performed with individual females were prepared using 1000 eggs in 1 ml hexane. Extracts for chromatographic analysis were prepared from 100 eggs in 100µl hexane for both L. renei and L. longipalpis and stored in sealed glass ampoules.

Attraction and/or oviposition stimulation bioassays: To test the biological activity of L. renei egg extracts, bioassays were carried out in plastic rearing pots (height 9.5 cm, diameter 15 cm) with plaster-of Paris lined bases. This experiment was an adaptation of the method used by Elnaiem et al. (1991). The base of the pot was divided in half and a semicircle of 4.5 cm radius was traced on either side of the dividing line, these being defined as the test and control areas. Filter paper discs (2.5 cm diameter), secured by a pin were placed within the semicircles . The egg extract (either 100 or 200 eggs/100µl) was applied to the test filter paper and the control disc was treated with 100µl hexane. Twenty gravid L. renei females were introduced into the assay pots 10 min after application of the test and control solutions. The insects were supplied with honey: water on cotton wool ad libitum and maintained in total darkness inside an incubator (25-26ºC; relative humidity 90%) for 48h. The numbers of eggs laid within the test and control sites were then counted.

The experiment was repeated 12 times for each treatment (extracts of 100 or 200 eggs). On completion of each replicate the plaster bases were changed and the pots and plastic boxes were washed in boiling water and 70% alcohol, rinsed six times in water and dried with paper towels and clean cotton.

Cage attraction bioassay: Acrylic cages (40 cm x 40 cm x 40 cm) were used to test the attractiveness of the egg extracts to gravid L. renei females. This method was an adaptation of that used by Dougherty et al. (1993). The base of the cage was lined with filter paper moistened with distilled water to ensure high humidity during the experiment. A filter paper disc (diameter 2.5 cm) was placed on an aluminum foil disc (diameter 4 cm) and then placed in the center of a 10 cm diameter Petri dish coated with an odorless glue (Tanglefoot Company, USA). Two sets of apparatus (test and control) were prepared. Each filter paper disk was then treated with either the test extract (100 or 200 eggs/100µl) or pure solvent control (100µl). The test and control dishes were marked and positioned in diagonally opposite corners of the interior of the cage, separated by a distance of 30 cm. After application of the test extract and the solvent control (100µl), 20 gravid L. renei females were introduced to the cage. Honey:water was provided in the upper central part of the cage. The experiment was carried out in the sand fly insectary (25ºC-26ºC; 80%-90% RH). The cage was covered with a black cloth left in place from 17:30-08:30. After this period the females found trapped in the Tanglefoot of the test and control dishes were counted. Twelve replicates of experiments using extracts of 100 eggs/100µl and eight of those using 200 eggs/100µl were done. The relative positions of the test and control dishes were changed among the four corners of the cage for each replicate to avoid positional bias. After each experiment the cage was cleaned with 70% alcohol and the black cloth washed with neutral soap.

Table 1. Attraction of gravid females of L. renei to extracts of 100 and 200 conspecific eggs in
100µl hexane in bioassays carried out in acrylic cages.

NS: differences not significant (t-test, P >0.05).P: probability level. %: Percentage of females captured.
^a :12 replicates: total 240 females. ^b 8 replicas: total 160 females.

Olfactometer attraction bioassay: In this bioassay an olfactometer was used to test the attraction of L. renei females to egg extracts. The olfactometer consisted of three rearing pots (11.0 cm diameter x 7.5 cm height) (Nalgene^®, UK) linked by two plastic tubes (9.5 cm long x 3.5 cm diameter (Nunc International^®, USA). The bases of the pots (designated as test and control chambers) were perforated and filled with plaster of Paris. Discs of filter paper (2.5 cm diameter) were secured by pins to the centers of the bases of the two lateral pots . Egg extracts (100, 200 or 1000 eggs/100µl hexane) were applied to the test discs and 100µl of hexane added to the control discs. Ten minutes after application of the extracts, 20 gravid females were introduced into the center pot and the entire assembly was placed inside a plastic box (56.4 X 38.5 X  20.1 cm, San Remo^®, São Paulo ), that was maintained in darkness inside an incubator (25-26ºC and 90% RH). After 24h the numbers of females and eggs found in the test and control chambers were counted. Twelve replicates of the 100 egg extract, 14 replicates of the 200 egg extract and six replicates of the 1000 egg extract were done. After each replicate was completed, the plaster bases of the pots were changed and the entire apparatus was washed in boiling water and 70% alcohol, rinsed six times in water and dried with paper towels and clean cotton.

Chemical analysis: Extracts of 100 1-2 day-old eggs/100µl hexane of L. renei and L. longipalpis from Lapinha Caves and their respective controls were analyzed by gas chromatography coupled mass spectrometry (GC/MS) (Dougherty and Hamilton, 1997). Peak area was used to compare the amounts of fatty acid found in L. renei with L. longipalpis eggs.

Analysis of results: The Kolmogorov-Smirnov statistical test was used to test for a normal distribution of each set of results. Those results that were normally distributed were compared by Student's t-test, the Wilcoxon and Kruskal-Wallis tests were used for those that did not. The 0.05 significance level was used.

Results

Attraction and/or oviposition stimulation bioassays: There was no significant difference between the number of eggs laid on the test and control sites when extracts of either 100 or 200 eggs were applied to test filter paper. When extracts of 100 eggs were applied the number of eggs laid on the test filter paper was 75.83±46.16 and on the control, 70.16±31.05. When extracts of 200-eggs were used, the mean number of eggs laid on the test filter paper was 50.33±39.35 and on the control was 45.25±33.36.

Cage attraction bioassay: The results of the attraction bioassays are shown in Table 1. In both bioassays (with 100 and 200 egg extracts) there was no significant difference between the number of females caught in the test and control dishes.

Olfactometer attraction bioassay: The results of olfactometer attraction bioassays are presented in Table 2. When 1000 egg extracts were placed in the test side of the olfactometer significantly more gravid females were attracted on average to the test side (7.66±3.07) than the control (3.66±2.73) (P=0.039). Also the mean number of eggs laid in the test chamber (139.33±62.48) was significantly higher than in the control chamber (68.16±41.71) (P = 0.043). When we tested 100 and 200 egg extracts, no significant difference was found between test and control results for either numbers of females attracted or numbers of eggs laid.

Table 2. Attraction of gravid females of L. renei to extracts of conspecific eggs in bioassays
carried out in the olfactometer.

^aDifferences not significant between mean no. eggs laid and mean no. females found in test and control
sites (t-test, P>0.05 ). ^b Differences significant between mean no. of eggs laid and mean no. females
found in test and control sites (t-test , P = 0.039 and P = 0.043 respectively). ¹ Total of 240 females
used in 12 replicates. ² Total of 280 females used in 14 replicates. ³ Total of 120 females used in 6 replicates.

Chemical analysis: In both species five different fatty acids (saturated and unsaturated) were encountered. The saturated compounds included dodecanoic, tetradecanoic and hexadecanoic acid while the unsaturated ones were represented by hexadecenoic and octadecenoic acid. In L. longipalpis dodecanoic acid was the most abundant fatty acid and this compound has been identified as the oviposition pheromone of this species (Dougherty & Hamilton 1997). Dodecanoic acid was found in smaller quantities in L. renei and octadecenoic acid was the most abundant compound.

Discussion

The stimulant effect of the extracts of conspecific eggs in L. longipalpis has already been demonstrated. The females individually exposed to the extracts laid a significantly higher number of eggs and a synergistic effect was observed when these extracts were combined with extracts of rabbit chow, post-oviposition survival increasing 3.5 times and oviposition in the test group being 2.5 times greater than that of the control group (Dougherty et al. 1993, 1994).

In the bioassays carried out in rearing pots, gravid females of L. longipalpis were clearly attracted to and/or stimulated to oviposit in the test sites where extracts equivalent to 100 or 160 conspecific eggs were applied (Elnaiem et al. 1991; Dougherty et al. 1992; Dougherty et al. 1993; Dougherty & Hamilton 1997). Females of P. papatasi responded in a similar manner, being attracted to/or stimulated to oviposit by the presence of 100 or 200 conspecific eggs (Srinivasan et al. 1995). However, in the present study extracts equivalent to 100 or 200 eggs did not produce attractive or stimulant effects on gravid females of L. renei, although a similar methodology was used we only tested sylvatic L. renei and all experiments with L. longipalpis or P. papatasi were from close colonies. Attempts were made to demonstrate a possible attractive effect of egg extracts on gravid females in the bioassays carried out in acrylic cages and the olfactometer. For bioassays of this nature to present trustworthy results, at least 50% of the total number of insects liberated should be recaptured in the test or control sites (RP Brazil, unpublished data). Although this threshold was reached in our experiments, no attractive effect was observed either for extracts of 100 or 200 eggs. An attractive effect was very evident for L. longipalpis when combined extracts of 100 eggs and rabbit chow were tested in similar bioassays, 62% of the total number of insects liberated being captured at the test site (Dougherty et al. 1993).

With regard to the bioassays carried out in the olfactometer, although at least 50% of the sand flies moved away from the central arena, no attractive effect was observed for females of L. renei when extracts of 100 or 200 eggs were applied to the test site, neither based on egg counts or the number of females. However, a significant response was observed for the gravid females of L. renei when extracts equivalent to 1000 eggs were utilized, with respect to both of these parameters. Counting the number of eggs alone may not be the most sensitive method of interpreting attraction to a stimulus (Isoe et al. 1995). Many female sand flies may not manage to lay eggs or die during oviposition, so that the number of eggs at the site does not necessarily indicate the number of females present. Counting females appears to be a more appropriate way of measuring the attractive stimulus in this type of assay.

Although in other sand fly species the response threshold was found to be close to the pheromone concentration present in 80 or 100 eggs (Elnaiem & Ward 1991, Dougherty & Hamilton 1997, Srinivasan et al. 1995), L. renei did not respond to quantities of extract equivalent to 100 or 200 eggs, at least in the type of bioassay used in the present study. Since no experiments were carried out utilizing extracts equivalent to more than 200 and less than 1000 eggs, we cannot determine the precise value of the response threshold to oviposition pheromone for gravid females of L. renei. Nor can we reject the possibility that gravid females of L. renei responded positively only to the extracts with large number of eggs because the oviposition pheromone was produced in abnormally low quantities by the insects in our study. This low production may to be related to inadequacy of the bloodmeal source utilized, it being believed that the precursors of oviposition pheromone are derived from the blood of the vertebrate host (Dougherty & Hamilton 1997).

Only the attractive effect of 1000-egg extracts in the olfactometer bioassays was demonstrated during the present study. Although the females in the test chamber laid a significantly higher number greater of eggs than those in the control, we cannot affirm that a stimulant effect was present, since the insects were not examined individually and it was not possible to determine how many oviposited, the number of eggs laid/female or the post-oviposition survival.

It was not possible to determine which of the fatty acids encountered on the surface of the eggs of L. renei were involved in semiochemical activity. This would require further studies using electroantennography and olfatometry to evaluate the activity of each substance encountered separately. The unsaturated fatty acids hexadecenoic and octadecenoic acid are more unstable than the saturated compounds and are therefore less likely to act as pheromones (JGC Hamilton, personal communication.). Among the saturated compounds, dodecanoic acid (the oviposition pheromone of L. longipalpis) and tetradecanoic acid were encountered in smaller quantities on the surfaces of the eggs of L. renei than on those of L. longipalpis. Although hexadecanoic acid was 1.5 times more abundant, it is important to emphasize that the compound present in greatest quantity is not always that responsible for semiochemical activity. It is also possible that the oviposition pheromone of L. renei consists of a mixture of compounds rather than a single substance as in L. longipalpis (Dougherty & Hamilton 1997). In the future new bioassays should be realized using L. renei from a close colony and not with sand flies collected in the field.

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