Studies on mosquitoes ( Diptera : Culicidae ) and anthropic environment . 8-Survey of adult behaviour of Spissipes Section species of Culex ( Melanoconion ) in South-Eastern Brazil

A study of Spissipes Section of Culex (Melanoconion) adults behaviour was carried out from August 1992 through December 1993 in human dominated (anthropic) environment in the Ribeira Valley, S.Paulo State, Brazil. By sampling at several sites it the dominance of Culex ribeirensis and Cx. sacchettae became evident even through a total number of ten species was recorded. Those two mosquitoes showed a clear tendency to frequent the domiciliary environment where they were caught, both indoor and outdoor, through the use of the human bait. In the outside environments, the residual patchy forests seems to display a concentration role, from which these adults spread to the open land and reach the dwellings. As their vector competence has been demonstrated through the virus isolations in natural conditions, it is advisable to pay attention to the presence of these mosquitoes in the man-made environment.


Introduction
As it is well known Culex (Melanoconion) are nocturnal mosquitoes of the Americas, inhabiting mainly forest environments and most active at dusk and dawn.They feed on the blood of vertebrates including humans, when available.Their competence to transmit various arboviruses has been demonstrated both through isolations from naturally infected mosquitoes and laboratory transmission trials.Thus, they are considered as important vectors of arboviruses in their sylvan natural cycle, specially the group of the Spissipes Section.Among these agents, the encephalitis viruses transmission has been seen as deserving of special treatment.
The southern geographical distribution of Melanoconion mosquitoes corresponds to the southern Brazil and northern Argentina.As far as is known, the Spissipes Section is there represented by eleven widely species spread.Five of these were found naturally infected with arboviruses.In Argentina, several bunyaviruses have been isolated from Culex delpontei and Cx.ocossa besides alphaviruses represented by strains of the Venezuelan equine encephalitis (VEE) and the western equine encephalitis (WEE) viruses (Calisher 3 , 1985; Mitchell et al. 20,21 , 1985, 1987).In S. Paulo State, Southern Brazil, strains of VEE and a number of bunyaviruses have been isolated from Cx. ribeirensis (formerly confounded with Cx. crybda and Cx.epanastasis), Cx. sacchettae and Culex sp.VR (hitherto misidentified as Cx.taeniopus) (Calisher et al. 1,2 , 1982, 1983;  Sallum 24 , 1994).Thus, the proportion of the isolations from these Melanoconion mosquitoes presages the incrimination species of that Section as important vectors of arboviruses.
During the undertaking of an investigation program in the Ribeira Valley region of S. Paulo State, mosquito behaviour was observed.In that area an encephalitis epidemic started earlier in 1975-1976 thereafter decreasing gradually until 1983 (Iversson 19 , 1988).Among others, mosquitoes belonging to the species referred above were focused mainly as regards their biting activities, peridomiciliary habits and its occurrence in primitive and residual woods in an anthropic environment (Forattini et al. 4,5,6,9,10 , 1981, 1986, 1990,  1991).The results of these observations were that Cx. ribeirensis and Cx.sacchettae appeared to be widespread in the region while Cx.delpontei, Cx. ocossa and Culex VR appeared to the limited to certain geographical areas, at least where they were found in larger numbers.Nevertheless with regard to the general anthropic and the particular dwelling environments, the two former species seems better adapted and quite attracted to human bait.
During the 1992-1993 period, a continuation of the above-mentioned investigation program, adult mosquito collections were performed through the use of human bait and the Shannon type traps.Considering the epidemiological importance of the Culex (Melanoconion) species the results of the research will be presented in this paper for the purpose of contributing to knowledge about their biting behavior in the anthropic environment.

Study areas
The data here reported are the results of sampling carried out at several sites on the Experimental Station (ES) and at the "Galiléia Farm" (GA) of the Pariquera-Açu County, S. Paulo State, Brazil, which were already described elsewhere (Forattini et al. 12,15 , 1993, 1994).According to the criteria adopted to mapping world ecosystems these sites were recognized as corresponding to disturbed environments (Hannah et al. 18 , 1994).The ES was included in the human dominated (anthropic) type where the agriculture is permanent and prevails over the primary vegetation that remains as limited remnants patches.As regards the GA environment, it was defined as partially disturbed, with secondary vegetation without intensive agricultural activity or any artificial irrigation system.The domiciliary environment was represented by the rural dwellings of the "Sitio Barra do Capinzal" (BC) that is included within the ES area.

Material and Method
Sampling was done in accordance with the procedures described elsewhere (Forattini et al. 15 ,  1994).Thus Shannon traps operated by a two-man team were used fortnightly from 17:00 to 20:00 hours at ES and GA sites, as follows: As Shan 1 and Shan 2 traps operated alternat, the total number of mosquitoes caught corresponded to the fortnightly rhythm.
Sampling the biting mosquitoes catches made in the domiciliary environment was done using human bait.Two collectors volunteers vacinated against yellow fever caught the females as they came to feed and alighted on their exposed legs during two hour crepuscular period, from 17:30 to 19:30 or from 18:00 to 20:00 hours according to the season (Forattini et al. 4,11,15 , 1981, 1993, 1994).The collections were simultaneously performed indoors and outdoors, by one man in each place using a batteryoperated aspirators to catch landing/biting females.Catches were made at BC from August 1992 through December 1993, following the fortnightly rhythm in a regular manner.The biting activity was estimated by the Williams media (x w was originally used (Haddow 16,17 ,  1954, 1960).
Regarding the degree of domiciliation, the synanthropic index of Nuorteva was calculated for both species, according to the percentages of specimens collected at the different places.In this manner an estimation of the synanthropy of these mosquitoes was proposed (Nuorteva 22 , 1963; Povolny 23 , 1971; Forattini et al. 13 , 1993).For this purpose the various collection places were considered as follows: Where: a = percentage collected in the domiciliary environment (BC).b = percentage collected in the anthropic and partially disturbed environments (Shan E and Shan GA).c = percentage collected in the remnant patchy forest environments (Shan 1 and Shan 2).s = Nuorteva index.
Thus the comparisons were undertaken within the disturbed anthropic environment and covered its various aspects here studied.To obtain a general over-view of Cx. ribeirensis's and Cx.sacchettae's degrees of synanthropy, the relations between the total numbers of specimens collected at each of the places referred to above were calculated as synanthropic ratios (sr).

Results
A total number of 6,631 adult mosquitoes (6,344 females and 287 males) of the Spissipes Section was collected.Of that number 5,602 (84.5%) were caught with the Shannon traps whereas 764 (11.5%) with the use of human bait.Ten species were obtained and their specific distribution was as follows: The results are presented, in accordance with the several samples obtained at the ES and GA sites, and the methods employed, in Table 1.Thus Cx. ribeirensis and Cx.sacchettae jointly accounted for 95.9% of the Spissipes Section specimens collected.The behavior of these two species called for particular attention in the present paper for that reason.
Shannon traps.-Themonthly distributions of Cx. ribeirensis and Cx.sacchettae are presented in Tables 2 and 3.Only females were obtained by the Shan GA.Considering the agricultural anthropic environment of the ES area it seems that those mosquitoes were mainly produced in the months of February-April when a total of 74.6% of the adults were caught.However, as a general feature, the remnant forest patches (Shan 1 and Shan 2) produced 85.1% of the total adults caught during the period, while 14.9% was obtained on the open land (Shan E).
In the partially disturbed environment of the GA site, the distribution pattern was quite irregular and no significant difference was observed.Domiciliary environment.-Throughthe use of human bait, a total of 758 females mosquitoes was caught of which 226(29.8%) belonged to Cx. ribeirensis and 532(70.2%) to Cx. sacchettae.
With respect to collection sites the results were as follows (Tables 1 and 4): The Williams' general means values were as follows: The individual values applied for the estimation of the Williams' means were compared by the Mann-Witney text, which showed the absence of differences, at the level of 5.0%, between Cx. ribeirensis and Cx.sacchettae.
Thus biting females of both species may be caught in the domiciliary environment, with similar frequencies (Table 4).As a general feature the peak of abundance corresponded to the December-April period when 540 (71.2% of the total) females were collected.That peak roughly corresponds to that obtained through the Shannon trap sampling (Table 2) in the ES area when 3,566 (74.2% of the total) females were collected during the February-April period.
Synanthropy -As has already been mentioned the three environments studied were compared by means of the synanthropic indices (s) for both the mosquito species collected in these habitats.The results obtained were as follows: Considering the general overview of this man-made environment, both mosquitoes appeared, in large measures, to retain their sylvatic habits.
Regarding the synanthropic ratios (sr) the overall value in the ES area, related to the remnant forest samples (Shan 1 and Shan 2) was 0.35 suggesting that these residual patches retain a greater number of both mosquitoes than the open land (Shan E) and the domiciliary environment (BC).Then, relating these latter two, the ratio obtained was 1.01, which shows no overall differences between these environments as regards the two culicids.Nevertheless, when the two species were considered separately the ratios obtained were the following: Thus, in the domiciliary environment (BC) Cx. sacchettae showed a higher synanthropy than Cx.ribeirensis when compared with the open land (Shan E) sampling.

Discussion
In a first attempt at an interpretation of the results obtained by the present research, the prevalence of Cx. ribeirensis and Cx.sacchettae among the Spissipes Section members must be considered.This fact was already noticed in previously published papers (Forattini et al. 5,6,13,14 ,  1986, 1993).Nevertheless this pattern must be taken as regional because in others regions of the Ribeira Valley others species may be prevalent.This happens in the Iguape area where Cx. delpontei, Cx. ocossa and Culex VR, jointly with Cx. ribeirensis and Cx.sacchettae, showed a high frequency in the amount collected (Forattini et al. 9 , 1990).
The main objective was to compare the adult abundance at the several sites.Two different techniques for sampling were employed, Shannon traps in the ouside environment and human bait in the domicilary one.This different aspects maybe influenced the results.However, according to the local circunstances, human bit was the more suitable sampling method that was used in dwellings.
The two residual patchy woods (Shan 1 and Shan 2) in the ES area accounted for the majority of the specimens of the two mosquito species caught.Jointly with the ES open land (Shan E), the monthly distribution showed a peak abundance corresponding to the February-April period.That corresponded to the results already reported (Forattini et al. 13 , 1993) for Cx.ribeirensis caught at the same place.When the two environments are compared it would seem that the remnant forest represents something like an area of concentration from which adults of the two culicids disperse and reach the open land, seeing only immature stages of Cx. ribeirensis, through as meaningless numbers, were found at the rice ES irrigation system (Forattini et al. 12 , 1993).
As regards biting behavior in the domiciliary environment, the general values of Williams 1 mean showed no difference as between the two species, December-April being the period of peak abundance roughly corresponding with that at the outside ES areas.Nevertheless within the same period secondary peaks of Cx. sacchettae were observed regarding that remarkably appeared in high numbers in the March samples (Table 4).It is hard to find an adequate explanation for this, but perhaps, besides the human bait, peculiar conditions related to the attractiveness of the increased number of fowl kept there may contribute to explain it.As regards Cx. ribeirensis, a more regular behavior in this environment was observed, in accordance with the general pattern already described for the natural outside environment (Forattini and Gomes 7 , 1988).
Regarding the degree of synanthropy of these two mosquitoes, both indices suggest weak tendencies to adapt to the domiciliary environment.This seemingly disagrees with the estimate in relation to the primitive environment for which indices of +63.0 for Cx.ribeirensis and -3.0 for Cx.sacchettae were obtained (Forattini et al. 13 ,   1993).Nevertheless the comparisons were made in the light of the human dominated environment as a whole.Indeed the anthropic one studied here included several different features each one with its own characteristics.Thus the remnant forests, the cultivated open land and the dwelling houses represente quite distinct aspects of the man-made organized environment.As was mentioned above the patches of residual forest may fulfil both the preserving and the concentration roles for these mosquito populations.So it is quite understandable that they retained their sylvatic behavior when compared with their behavior in others places.Despite the comparison of their synanthropic ratios (sr), it became clear that, relating the open land to the domiciliary environment, the two culicids showed a higher frequency in the latter.Cx. sacchettae at least seems to have preferred the houses over the open land.As a general aspect, both species have showed a clear tendency towards domiciliation (Forattini et al. 8 , 1989).
In conclusion, the present research permits to consider Cx. ribeirensis and Cx.sacchettae as potentially synanthropic, at least in the Ribeira Valley region.As their competence for virus transmission has been reported, it is justifiable to pay attention to these mosquitoes in the anthropic environment.