Effects of chickens on the prevalence of infestation and population density of Triatoma infestans in rural houses of north-west Argentina

Abstract Effects of the presence of chickens on population density of the bug Triatoma infestans , principal vector of Chagas disease, were investigated by standardized sampling (indoors and peridomestically) from sixty-eight houses of three rural communities in north-west Argentina, during March 1992.
The domiciliary density of T. infestans increased linearly with the percentage of bugs that fed on chickens, as identified by agar double-diffusion tests. Bug density was significantly higher in houses where hens usually nested indoors than in those where they did not, as determined by concurrent direct observations and interviews of householders. Multiple linear regression analysis of domiciliary bug density on (a) the total number of people, dogs and cats per house; (b) the percentage of domiciliary bugs that fed on chickens, or (c) the indoor-brooding habit of hens, showed the two variables (b, c) related to chickens as significant predictors in each regression model. Inclusion both variables representing chickens increased the fit significantly. Addition of other potentially confounding factors (domestic insecticide use, type of roof and walls of house) did not affect the significant variables retained in the best-fitting regression model.
Peridomestic infestation was positively associated with the household number of fowls. Chickens were the main bloodmeal source of peridomestic T.infestans populations. Human-fed bugs were detected in peridomestic sites of sixteen houses, indicating active dispersal of adults and large nymphal instars from bedroom areas.
Exclusion of hens from domiciliary areas and promotion of chicken sheds, of an appropriate design that would not harbour bug populations, should limit the triatomine population growth rate and reduce the risk of infestation.     

Population dynamics and control of Triatoma infestans

Analysis of field populations of Triatoma infestans (Hemiptera: Reduviidae), after a 3-year study, shows that population growth rate is affected by both density-dependent and density-independent mortality. Although an equilibrium exists, apparently as a consequence of a density dependent-mechanism, population density fluctuates throughout the year because of the effect of monthly mean minimum temperature as a density-independent source of mortality. Simulation studies based on Moran curves shows that high population densities have an approximately constant extinction probability (around 0.20), independently of the season the population starts growing. However, at very low population densities, the extinction probability depends strongly on the season when the population begins to grow. Very low density populations beginning in winter or autumn have the highest extinction probability. The outcome of the simulation studies coincides with results observed in field populations affected by insecticide application at different seasons.     

Determinants of the domiciliary density of Triatoma infestans, vector of Chagas disease

In two heavily infested rural villages of Santiago del Estero, Argentina, where no indoor-spraying with residual insecticides had ever been carried out by official control services, we studied the influence of roof and wall structure, domestic use of insecticide, family size and the number of domestic dogs, on the domiciliary density of Triatoma infestans (Klug). Bug density was significantly associated with (1) the interaction between insecticide use and type of roof, (2) the structure of indoor walls, (3) the number of dogs sharing sleeping areas of people (room-mate dogs), and (4) the number of people plus room-mate dogs, but not with just the number of people resident in the house. The interaction between insecticide use and a roof made of 'simbol', a locally available grass (Pennisetum sp.), also reflected a younger age structure of domestic bug populations. In infested houses, the density of bugs infected with Trypanosoma cruzi Chagas was significantly correlated with overall bug density. Our data suggest that the application of environmental management measures by the affected people, such as plastering of walls and modification of roofs, coupled with keeping dogs away from bedrooms and application of insecticides, should limit the domestic population density of T. infestans and thus reduce the transmission of T. cruzi to people.     

The biting rate of Triatoma infestans in Argentina

1. The daily proportion of fed individuals in a population of the reduviid bug Triatoma infestans (Klug), maintained under natural climatic conditions in experimental chicken houses in central Argentina, was estimated from the proportion of bugs retaining colourless urine in the rectum. 2. From the estimates of feeding frequency throughout a 1 year period, it was shown that temperature has a dominant effect on biting rate, but density-dependent effects became apparent during the warmest months. 3. These and other data on the determinants of blood consumption by T. infestans, were incorporated into a detailed hypothesis of density regulation in this species.     

Population dynamics of Triatoma infestans under natural climatic conditions in the Argentine Chaco

Using experimental chicken houses at a site in central Argentina where the bug Triatoma infestans (Klug) is endemic, nine populations of this vector of Chagas disease were monitored during a 34-month period. Bug populations with four chickens as hosts were consistently larger than those with two chickens as hosts. Age structure of the bug population followed a similar pattern irrespective of the initial age structure. Egg to adult mortality was consistently around 98.5% and there was no consistent evidence for density-dependent mortality. There was some evidence for density dependence in fecundity and recruitment rates, but these were heavily constrained by low temperatures during the winter months. Nymphal development rates correlated most strongly with mean minimum temperatures rather than with mean maximum temperatures. We conclude that vector control using insecticides against this species would be most effective at the onset of winter, when recovery of any surviving populations would be inhibited by low temperatures.     

Comparison of management strategies for squash bugs (Hemiptera: Coreidae) in watermelon

Two watermelon pest management practices, a squash trap crop and a standard recommendation using soil-applied carbofuran, were compared using large-scale field plots to assess trap crop suitability as a replacement for the standard in 2000, 2001, and 2002. In both systems, foliar insecticide applications were used to control squash bugs when populations exceeded threshold levels. During 2001 and 2002, a treatment of untreated watermelon was used. Early season adult insects, from seedling to fruit set, are most critical for watermelon. Significantly fewer early adult bugs were found on watermelon in the trap crop than in the standard recommended practice in 1 of 3 yr. In both years, significantly fewer adult squash bugs were found in watermelon in the trap crop than in untreated fields. The standard recommended practice significantly reduced adult squash bugs in watermelon compared with the untreated in 1 of 2 yr. There was no significant correlation of watermelon yield and squash bug density, indicating that squash bug densities were too low to impact yield. Although squash bugs were reduced significantly by the trap crop, marketable watermelon yields were lower in the squash trap crop than in untreated watermelon, suggesting that pest management treatments may interfere with crop productivity factors other than squash bug colonization. Results suggest that mid-season production squash bug should be managed by monitoring populations and using insecticides as needed rather than using at-plant treatment. Further research is needed to compare treatments during early-season production.     

Residual effect of a micro-encapsulated formulation of organophosphates and piriproxifen on the mortality of deltamethrin resistant Triatoma infestans populations in rural houses of the Bolivian Chaco region

The Bolivian Chaco is part of the endemic region of Chagas disease and an area where pyrethroid resistant Triatoma infestans (Hemiptera: Reduviidae) populations has been reported. The World Health Organization identified these resistant populations as an important focus for research. The objective of this study was to evaluate the residual effect of a micro-encapsulated formulation containing organophosphate active ingredients and a juvenile hormone analogue (Inesfly 5A IGR) on the mortality of T. infestans. Studies took place in rural houses of the Bolivian Chaco that were treated up to 34 months before and evaluated the susceptibility to pyrethroids of the offspring of field collected insects. Thirty houses were randomly selected within three communities to carry out wall bio-assays with T. infestans nymphs. Mortality was recorded 24, 48 and 72 h after wall contact. Eggs laid by females collected in the area were used to obtain first-instar nymphs and carry out pyrethroid susceptibility tests. The wall bio-assays showed that the micro-encapsulated insecticide eliminates T. infestans populations and produces detectable mortality of insects exposed to walls treated 34 months prior to the tests. The discriminant dose of deltamethrin (0.01 mg/mL) showed 65% nymph survival, whereas at the highest tested dose (1.0 mg/mL) 14% of the nymphs survived. These results show that Inesfly 5A IGR is an appropriate tool for the elimination of intradomestic and peridomestic populations of T. infestans resistant to pyrethroids.     

Comparison of two sampling methods for domestic populations of Triatoma infestans in north-west Argentina

Abstract. In mud-and-thatch houses of Santiago del Estero Province, northwest Argentina, where no insecticides had been sprayed officially to control domestic infestations of the Chagas disease vector Triatoma infestans (Klug) (Hemiptera: Reduviidae), two knockdown (KD) insecticidal collection procedures were compared with the standard flush-out (FO) method for sampling T. infestans. Bugs were collected by FO using 0.2% tetramethrin in bedrooms of (1) 41 houses of Amama village employing 1 man-hour of capture effort per house, and (2) 19 houses of Trinidad and Mercedes villages employing 4 man-hours/house. From the same houses, 2-5 days after the manual FO collection, bugs were collected by KD indoor-spraying of deltamethrin 25mga.i./m² in Amama, or burning of one γ-HCH (=γ-BHC) fumigant tablet 3.1g of γ-isomer) per bedroom in Trinidad and Mercedes. The majority of infestations were detected by both methods, the proportion of positive houses being 81% at Amama and 95% at the other villages. Although the FO method was more sensitive, at least because it was applied first, the KD method detected infestations in 25% of houses where bugs were not found by FO. Bug densities estimated by FO or by subsequent KD in each house were significantly correlated: r = 0.795 for deltamethrin; r= 0.882 for y-HCH. Compared with FO collections of T.infestans large stages, i.e. adults plus fourth and fifth instar nymphs, the KD catch averaged 0.88x with deltamethrin and 0.57× with γ-HCH, regardless of the apparent population density of bugs per house. However, the KD method has practical advantages of speed and standardization.     

Experimental manipulation of the timing of reproduction in the California vole

Summary The potential role of food and water in determining 1) the timing of the normal breeding season in the California vole, and 2) two frequently occurring exceptions to this normal pattern, are explored by using experimentally manipulated enclosed populations. Hypotheses derived from field observations were tested in the enclosed populations by the manipulation of green food, dry food, water, and vole density. The results of three experiments involving five populations are reported. In all populations densities were maintained at near-normal levels (475 to 1750 per ha). In all three experiments, it was shown that breeding was possible in the summer if sufficient food and water were available, although perhaps with reduced litter sizes and rates of growth and reproductive maturation of young. In two cases, severe weight losses and high mortality were induced by insufficient water. When water was provided, survivors recovered weight and reproductive competency in between five weeks and eight months. In three populations, starvation induced weight losses and mortality. In these cases recovery of the voles took only one month or less. In two of these last, the inadequate food situation was produced during the normally wet season by outbreaks of a European slug. It is concluded that available moisture is the most important factor in triggering both the end and beginning of the normal breeding season. Of course, inadequately fed mice do not breed at any season. Summer (dry season) breeding can occur when food and water conditions are adequate due to locally favorable conditions or very low population densities. Prolonged delays in growth and reproduction following the return of favorable conditions are most likely the consequence of chronic and severe water deprivation.     

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi in Triatoma infestans (Insecta, Hemiptera).

The effect of azadirachtin on Blastocrithidia triatomae and Trypanosoma cruzi, which colonise the intestinal tract of the blood-sucking bug Triatoma infestans, was investigated. In established infections of controls without azadirachtin treatment, the small intestine of fifth-instar T. infestans contained up to 7 x l0(6) B. triatomae and the rectum 3 x 10(6). In comparison to this homoxenous flagellate, the population densities of T. cruzi in the respective regions were 99.3 and 76% lower. Treatment with azadirachtin (1 microg ml(-1)) via a blood meal and a concurrent infection with B. triatomae resulted in an increase of the population density (3 weeks p.i.), caused mainly by the mastigote stages in the rectum. In an established B. triatomae infection (12 weeks p.i.), feeding of azadirachtin did not affect the population density and composition. In an optimal T. cruzi-vector system, i.e. parasite and bug originate from the same locality, the treatment with azadirachtin at 20 weeks p.i. strongly reduced the population density in the small intestine of all bugs up to 100 days after treatment, but only in a minor percentage of the bugs in the rectum. Trypanosoma (cruzi incubated for up to 24 h in faeces of azadirachtin-treated bugs were not affected, indicating that the rectum of these bugs contained no toxic substances. The importance of these findings is that investigations of the mechanisms of action of azadirachtin offer a possibility to identify vector-derived compounds, which are necessary for the development of T. cruzi, thereby, giving us a possible new strategy to combat Chagas' disease.     

Abundance and nightly activity behavior of a sylvan population of Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) from the Yucatan,México

Triatoma dimidiata is the vector of Trypanosoma cruzi in the Yucatan Peninsula (YP). Earlier studies have shown that domestic and peri‐domestic populations of the vector originated from the sylvan stock and that effectiveness of insecticide‐spraying was affected by re‐infestations of houses from the sylvan T. dimidiata population. In addition, in the YP most previously published reports have focused on domestic and peri‐domestic populations and very little is known about the nocturnal behavior of the sylvan populations. The main aim of our study was to determine the nightly activity patterns of adult T. dimidiata in a selected location in the YP. Secondly, we sought to document the reproductive status and infection rate of active females. During eight sampling nights spaced from late March to late July, 2007, we collected 544 adult T. dimidiata. We found that square‐cloth illuminated white traps were effective to attract the sylvan individuals and that T. dimidiata adults exhibited a unimodal activity pattern throughout the night. The accumulated mean of captured bugs also showed a non‐linear distribution for females and males. Furthermore, we found that male and female catches were significantly correlated with the means of temperature and humidity recorded during the sampling period. Out of 46 dissected females, we observed that 43.5% of females had fully‐formed eggs in their abdomens, and only two females (4.4%) had sperm within the spermatheca. The infection rate of T. dimidiata harboring T. cruzi was found to be 3.7%. The implications of the light attraction to bugs and potential dispersal capabilities are discussed in the paper in the context of infestation/re‐infestation of rural houses by sylvan T. dimidiata flying adults.     

Seasonal variations in active dispersal of natural populations of Triatoma infestans in rural north-western Argentina

The flight dispersal of Triatoma infestans Klug (Hemiptera: Reduviidae) is one of the main mechanisms determining community re-infestation after control interventions. An empirical model of flight initiation coupled with data from a longitudinal study predicted that the flight dispersal of T. infestans would peak in summer. To test this prediction, longitudinal light trap collections were conducted during 3-8 nights in March (late summer), July (winter) and November (spring) 2003, and in March 2004 in a rural community in north-west Argentina. Following each light-trapping collection date, all peridomestic sites around light traps were inspected to assess the relative abundance and nutritional status of T. infestans at each site. A total of 21 adult and five nymph T. infestans, six Triatoma guasayana Wygodzinsky & Abalos, and nine Triatoma garciabesi Carcavallo et al. were collected in 96 light-trapping nights, whereas 696 T. infestans were collected from the peridomestic sites that surrounded the light traps. The arrival of T. infestans in the light traps occurred in 64% of catch stations and peaked in the summer surveys (10-14 bugs) compared with spring and winter surveys. When winds were < 5 km/h, the arrival of adult T. infestans at the light traps was significantly associated with maximum temperature and relative humidity. This is the first field report of seasonal variations in the flight dispersal activity of T. infestans.     

Infestation of rural houses by Triatoma infestans in the region of Los Llanos (La Rioja, Argentina)

Vectorial transmission of Chagas disease has been strongly reduced in most parts of the Southern Cone countries of South America, except in the Gran Chaco region of Argentina, Bolivia, and Paraguay. Given periodical interruptions of the vector control programmes in the endemic region of the Gran Chaco of Argentina, the vectorial transmission of the disease has been increasing during the last years. From the beginning of 2004, the provincial Ministry of Health of La Rioja, Argentina, started a vector control programme to cover the rural houses of the Los Llanos area in the southwestern area of the Gran Chaco region. This article reports the result of a standardized entomological survey and insecticide application against Chagas disease vectors in the intra and peridomestic structures of the rural houses of Los Llanos. A total of 4062 houses were inspected, of which 46.8% were found to be infested by Triatoma infestans. Infestation by vector species other than T. infestans was less than 0.5%(T. eratyrusiformis and T. platensis). Intradomestic infestation was found in 27.2%, whereas peridomestic infestation was found in 39.3% of the houses. The lowest figure of intradomestic infestation was 6.6% (Department F Varela), and the highest value of intradomestic infestation was 45.1% (Department Independencia). In spite of the demonstrated success of vector control elsewhere, this study shows that the vector populations are susceptible to pyrethroid insecticides in the southern area of the Gran Chaco of Argentina, that there still are regions where rural houses show heavy infestation by T. infestans associated with big peridomestic structures and that the vectorial transmission of the Chagas disease will continue, unless a sustained and well organized vector control effort is installed in the region.     

The effect of plant density on populations of the cabbage root fly on four cruciferous crops

To study the effects of plant density on populations of the cabbage root fly (Erioischia brassicae), cabbage, cauliflower, Brussels sprout and swede were each planted in plots with twenty-four concentric circles of plants at spacings ranging from 10 to 90 cm between the individual plants. Plants treated with a root drench of chlorfenvinphos and untreated plants were each sampled at ten plant densities which ranged from 1–5 to 68-3 plants/m². In the absence of insecticide, the numbers of overwintering cabbage root fly pupae produced ranged from c. four per m² at the lowest plant density to 200 per m² at the highest. The number of pupae per m² was proportional to plant density to the powers 0–98,0-77,0–69 and o-6i for the swede, cauliflower, cabbage and Brussels sprout crops, respectively. The magnitude of each cabbage root fly population was determined mainly by plant density but also by the cultivar used as host plant. The results suggested that, in a given locality, when changing from low to high plant density crops during a growing season it should be unnecessary to apply insecticide to control cabbage root fly; conversely, a change from high to low plant densities would necessitate an extremely efficient application of insecticide.     

Macrogeographic and microgeographic genetic structure of the Chagas’ disease vector <Emphasis Type="Italic">Triatoma infestans</Emphasis> (Hemiptera: Reduviidae) from Catamarca,Argentina

The genetic structure in populations of the Chagas' disease vector Triatoma infestans from six localities belonging to areas under the same insecticide treatment conditions of Catamarca province (Argentina) was examined at macrogeographical and microgeographical scales. A total of 238 insects were typed for 10 polymorphic microsatellite loci. The average observed and expected heterozygosities ranged from 0.319 to 0.549 and from 0.389 to 0.689, respectively. The present results confirm that populations of T. infestans are highly structured. Spatial genetic structure was detectable at macrogeographical and microgeographical levels. Comparisons of the levels of genetic variability between two temporal samples were carried out to assess the impact of the insecticide treatment. The genetic diversity of the population was not significantly affected after insecticide use since different genetic parameters (allele number, observed and expected heterozygosities) remained stable. However, loss of low frequency alleles and not previously found alleles were detected. The effective population size (N(e)) estimated was substantially lower in the second temporal sample than in the first; nevertheless, it is possible that the size of the remnant population after insecticide treatment was still large enough to retain the genetic diversity. Very few individuals did not belong to the local T. infestans populations as determined by assignment analyses, suggesting a low level of immigration in the population. The results of the assignment and first-generation migrant tests suggest male-biased dispersal at microgeographical level.     

Effect of deltamethrin spraying on the sandfly populations in a focus of American cutaneous leishmaniasis.

The effect of deltamethrin of the sandfly population in a focus of American Cutaneous Leishmaniasis in Viana, Espírito Santo State is described. The phlebotomine population density was determined inside and outside houses during a one year period in a treated and untreated area. The results showed a significant reduction in the number of sandflies inside houses in the treated area compared with both the untreated area and the same area before spraying. Despite having a residual action for 12 months after spraying the insecticide was ineffective outside houses. It is concluded therefore that deltamethrin is an efficient insecticide for reducing the number of phlebotomine sandflies inside houses.     

Seasonal variation in benthic invertebrate recolonization of small-scale disturbances in a subtropical Florida lake

Approximately 11.5% of the littoral zone of a hypereutrophic Florida lake is disturbed by the construction of spawning beds by the cichlid fish, Sarotherodon aurea, during a single spawning season. Simulations of these beds were created during three seasons of the year to test for variation in recolonization rates and patterns in the benthic macroinvertebrate community.The seasonal variation in densities of benthic invertebrate populations suggests a direct relationship between reproductive activity and recolonization rate. Recolonization rates of the numerically dominant species, Polypedilum halterale (Diptera: Chironomidae), were much more rapid in the spring and summer than in the winter. In contrast, Hyalella azteca and Glyptotendipes paripes attained peak population densities during the winter season when Polypedilum was present in low densities. These organisms invaded the disturbed area in unusually high abundances and later declined to background levels. The final result was a winter population with densities comparable to the control (undisturbed) area, but the species composition was different.Similarity between disturbed and control communities during the winter season only reached 67% a month after disturbance, while communities during the warmer months attained nearly 90% similarity in less than 15 days. This lack of similarity during the winter indicates that disturbance, at the appropriate time, may play a role in community organization.     

蚜虱净对苜蓿主要害虫及天敌种群数量的影响

在甘肃省定西市九华沟系统研究了喷施蚜虱净对苜蓿主要害虫及天敌种群数量动态的影响。结果表明,施药田蚜虫和蓟马的季节平均数量极显著高于对照田,而盲蝽和瓢虫、小花蝽、蜘蛛等大多数天敌的季节平均数量则极显著低于对照田,寄生蜂与对照田没有湿著差异。喷施蚜虱净对种群数量时序动态的影响在不同害虫和天敌种类中表现不同,5月底施药后7d对照田蚜虫和蓟马的种群数量分别为施药田的4．13倍和5．2倍,此后施药田害虫的种群数量开始迅速增加,到6月底2种害虫的种群数量均与对照田无显著差异,到7月中旬以后反而显著高于对照田。施药对盲蝽种群数量时序动态的影响与蚜虫和蓟马不同,5月底施药后盲蝽的种群数量一直显著低于对照田,直到7月下旬以后,种群数量才恢复到对照田的水平。喷施蚜虱净对天敌种群数量的时序动态有极显著的影响,大多数天敌的种群数量在5月底施药后到7月底都一直显著低于对照田,直到8月上旬才恢复到对照田的水平。施用杀虫剂在防治害虫的同时杀伤大量天敌,使害虫的种群数量迅速增加,甚至造成更严重的为害。因此在菖蓿上使用对天敌杀伤力小的杀虫剂对保持天敌的自然控制能力有重要作用。     

Comparative use of bendiocarb and DDT to control Anopheles pseudopuncitpennis in a malarious area of Mexico

The state of Sinaloa has one of the highest and most persistent malaria transmission levels in Mexico. Due to this situation, with resistance of the vector Anopheles pseudopunctipennis Theobald to DDT, the carbamate insecticide bendiocarb was evaluated as an alternative to DDT for residual house-spraying in village-scale trials during 1985-87. Application rates of the active ingredient per square metre of sprayable surface (ai/m2) were 0.4 g bendiocarb 80% wettable powder (80WP) and 2 g DDT 75% WP. Both insecticides failed to control mosquito populations. Human-bait mosquito densities were not altered as a result of insecticide spraying and human-bait collected mosquito mortality rates were low, suggesting little pre-biting insecticide contact due to avoidance or insufficient resting time indoors. Lower densities of indoor-resting mosquitoes were observed with DDT as opposed to bendiocarb treated houses. Anopheline mortality was higher (98-100%) when exposed for 1 h to 1% bendiocarb in standard WHO susceptibility tests and wall bioassays. Mortality-rates of 15-48% due to 1 h exposure to 4% DDT indicated that this insecticide may continue to be partially effective. House curtain and mark-recapture mosquito studies indicated that DDT produced higher excito-repellency than bendiocarb, as reflected by more mosquito landings but lower feeding rates, shorter resting period and earlier exit time from DDT sprayed houses. In the absence of insecticide, more than 50% of blood-fed An.pseudopunctipennis females exited from houses within 2-4 h of release, showing exophilic behaviour. The outdoor/indoor density ratio indicated that the majority were exophagic. These behavioural characteristics limit the usefulness of any residual insecticide against An.pseudopunctipennis.     

Molecular Population Genetics and Evolution of the Chagas’ Disease Vector Triatoma infestans (Hemiptera: Reduviidae)

Triatoma infestans (Klug) is the main vector of Chagas’ disease in the Southern Cone of Latin America between the latitudes 10° S and 46° S. The long-term effectiveness of the control campaigns is greatly dependent upon the vector population structure. Mitochondrial DNA (mtDNA) genes have been used in a number of T. infestans population genetic analyses. However, the maternally inherited markers as well as nuclear ribosomal DNA analyzed until the present exhibited low or limited levels of variation. Analyses based on microsatellite markers strongly supported the existence of some type of stratification in T. infestans populations and supported the hypothesis of vector population recovery from survivors of the insecticide-treated areas, highlighting the value of population genetic analyses in assessing the effectiveness of Chagas’ disease vector control programmes. Although phylogeographic studies have generally suggested a Bolivian Andean origin of T. infestans, they recovered two reciprocal monophyletic groups of T. infestans and Bolivian populations who were not basal as expected for an ancestral group. In addition, a non-Andean origin could not be excluded by mtDNA genealogies that included sylvatic bugs from Gran Chaco. On the other side, mitochondrial and microsatellite markers supported the hypothesis of two independent migration events of colonization and secondary contacts in southern South America. Since the phylogenetic analyses remain inconclusive, more sequences, not only from mitochondrial genes but also from nuclear genes, need to be examined.