Anopheles belenrae Rueda

Rueda, Leopoldo M., Brown, Tracy L., Kim, Heung-Chul, Klein, Terry A., Thongkukiatkul, Amporn & Sherwood, Van, 2009, Description and comparison of morphological structures of the eggs of Anopheles hyrcanus group and related species (Diptera: Culicidae) from the Republic of Korea, Zootaxa 2268, pp. 23-40 : 34-38

8. Anopheles belenrae Rueda

( Figs. 3 H, 4H, 5H, 6H)

Size: Length 518.76–604.87 um (mean 572.07 + 40.44 um, n = 5); width 66.51–150.62 (mean 129.09 + 40.65 um, n = 5) (Table 1). Color: Black. Overall appearance: Boat-shaped in both ventral and dorsal views, anterior end blunt, posterior end blunt, sometimes pointed. Ventral surface concave, dorsal surface curved, float relatively short and wide in dorso-ventral plane, length 266.87–304.33 um (mean 293.80 + 15.59 um, n = 5); width 57.86–111.08 um (mean 74.44 + 22.25 um, n = 5). Dorsal and lateral surfaces: All surfaces uniformly covered with mostly pentagonal and hexagonal outer chorionic cells or plastron-type cells ( Hinton 1968) ( Fig. 3 H), each longer than wide, long dimension oriented in long axis of egg. Interior of each cell with interconnected, fine rounded structures, surrounded by an elevated, palisade-like outer chorionic reticulum. Cell area 98.38–374.86 um (mean 264.84 + 66.08, n = 14) (Table 1). Float fairly short, about 0.51 length of egg; ratio of float length and width, and length in proportion to egg length and number of ribs as in Table 1. Ribs towards both ends of float wider than those at middle part, rarely striated on dorsal sides; number of ribs per float 21–28 (mean 23.63 + 2.20, n = 8). Ventral surface. Deck continuous, narrows in mid-line near center of float, degree of narrowing usually variable; anterior part of deck usually wider than posterior part; entire deck covered uniformly with fine tubercles ( Fig. 4 H). Frill continuous, shallow along narrowed portion of deck. Lobed ventral tubercles at anterior end of the deck, 4–9 (mean 7.75 + 2.50, n = 4), and at posterior end, 4–7 (mean 5.00 + 1.73, n = 3) (Table 1, Fig. 5 H). Lobed ventral tubercles usually oval or oblong, occasionally round. Lobes of each anterior ventral tubercle, 4–10 (mean 7.50 + 1.41, n = 22; lobes of each posterior ventral tubercle, 3–4 (mean 3.33 + 0.58, n = 3). Lobes clearly separated, often swollen at ends, outer walls often smooth. Lobes in slightly elevated, tuberculoid structures. Anterior end, micropyle. Anterior end slightly more blunt than posterior end. Micropylar collar irregular in outline, with slightly striated surface, inner edge uniformly and deeply excavated, peaks between excavations tapering to form radial ridges extending about half way across micropylar disc, dividing disc into sectors ( Fig. 6 H). Number of sectors (or ridges) 6–8 (mean 7.25 +0.96, n = 4). Area of micropylar disc 20.42–21.40 um (mean 20.98 + 0.50 um, n = 3), usually with striated surface.

Morphological comparisons. Table 1 shows the comparisons of 27 attributes of eggs among eight Anopheles species, (see Appendix for abbreviations and their equivalents). Four species (i.e. An. koreicus , An. lesteri , An. lindesayi japonicus , An. sineroides ) have significantly higher float/egg length ratios, from 0.72– 0.76, compared with the remaining four species (from 0.51–0.57) (P <0.05). Anopheles koreicus has a very distinct thin ornamented layer, covering the entire deck. This layer has elongate openings on both ends that extend towards the middle of the deck. It has also a usual round opening at middle part of the deck. Anopheles belenrae eggs are significantly longer than those of An. kleini , An. koreicus , An. lesteri , and An. sinensis (P <0.05). Anopheles sinensis eggs have significantly wider middle decks than those of An. belenrae , An. kleini , An. koreicus , and An. sineroides (P <0.05).

Principal Components. Principal components are useful as a means of identifying the combinations of attributes that provide the important contrasts and differences among usable characters of various species. Six attributes derived from SEM micrographs of the eggs were used: EGGL, EGGW, FLWR, FLELP, FLOA, FLOL (see the Appendix). They were selected using cross tabulations, correlations, parametric statistics and principal component analysis (PROC ANOVA, PROC PRINCOMP; SAS Institute 2003). Of the six derived from the standardized (zero, mean, unit variance), variables, the first three accounted for 89.28% of the variation, and the first two for 70.34% ( Table 2). Component 1 carried a heavy positive weighting ( Fig. 7 ) for maximum length of float (FLOL). The attribute egg width (EGGW) had a negative eigenvector in component 1 ( Table 2). Component 2 accounted for about 50% less than component 1 ( Table 2). Attribute egg width (EGGW) contributed strongly to weightings on this axis ( Fig. 8 ). Component 3 accounted for 18.94% of total variance. The heaviest weightings in this component were mostly egg width (EGGW) and float area (FLOA), although length/width ratio of the float (FLWR) yielded a heavy negative weighting in component 3 ( Table 2).

Principal Eigenvalue % of variance Attribute*

component explained EGGL EGGW FLWR FLELP FLOA FLOL 1 2.7397 45.66 0.1516 -0.1428 0.0509 0.5577 0.5495 0.5839 2 1.4807 24.68 0.6762 0.5702 0.3795 -0.1899 -0.0716 0.1795 3 1.1364 18.94 0.1733 0.3661 -0.8214 -0.1449 0.3638 -0.0878

*Attribute defined in the Appendix.

Discriminant Functions. When discriminant analysis was applied to six variables to facilitate the separation of the species, the first four functions proved to be significant and the first 2 have 82.75% of the differences among species ( Table 3). Four species ( An. belenrae , An. kleini , An. pullus and An. sinensis ) were centered on the negative side of the first discriminant function, whereas the four other species were positive ( Figs. 9 and 10 ). For the second discriminant function, an equal number of species appeared on either side of the centerline, but An. koreicus was far removed from the others. Examining the centroids, species differences are found primarily between a group (composed of An. sineroides , An. lesteri and An. lindesayi japonicus ), An. koreicus , and another group (composed of An. kleini , An. sinensis , An. pullus and An. belenrae ) ( Fig. 10 ).

Although eggs of Anopheles in South Korea are relatively simple in structure, they differ clearly in some attributes at the stereomicroscopic level (Table 1), particularly An. koreicus . Multivariate analysis of egg characters indicated that An. kleini , An. sinensis , An. pullus and An. belenrae generally cluster together ( Figs. 8 and 10 ), and that their separation from An. lesteri , An. sinensis , and An. lindesayi japonicus on the second principal component was primarily attributable to differences in egg width. Several studies (e.g. Linley 1992, Linley et al. 1993, Linley et al. 1996) used principal component analyses to separate eggs of different Anopheles species from different locations. Further studies need to be done to compare morphological differences of mosquito eggs from separate geographical populations of Anopheles vectors of malaria, not only from the ROK, but also from other countries. These data maybe useful in revising the taxonomy of various Anopheles groups.

Discriminant function Eigenvalue Relative percentage Chi-squared df P 1 5.4907 67.93 147.37 42 0.0000 2 1.7890 14.82 81.90 30 0.0000 3 0.7901 6.72 46.01 20 0.0008 4 0.5227 6.09 25.63 12 0.0121 *Attribute defined in the Appendix.

Figures

FIGURE 3. Anterior dorsal plastron of Anopheles (Anopheles) species. A, An. kleini; B, An. sinensis; C, An. sineroides; D, An. pullus; E, An. lindesayi japonicus; F, An. koreicus; G, An. lesteri; H, An. belenrae. Magnification, 3500 x.

FIGURE 4. Chronic tubercles of middle part of deck of Anopheles (Anopheles) species. A, An. kleini; B, An. sinensis; C, An. sineroides; D, An. pullus; E, An. lindesayi japonicus; F, An. koreicus; G, An. lesteri; H, An. belenrae. Magnification, 3500 x.

FIGURE 5. Anterior ventral tubercles of Anopheles (Anopheles) species. A, An. kleini; B, An. sinensis; C, An. sineroides; D, An. pullus; E, An. lindesayi japonicus; F, An. koreicus; G, An. lesteri; H, An. belenrae. Magnification, 3500 x.

FIGURE 6. Dorsal micropylar discs of Anopheles (Anopheles) species. A, An. kleini; B, An. sinensis; C, An. sineroides; D, An. pullus; E, An. lindesayi japonicus, F, An. koreicus, G, An. lesteri, H, An. belenrae. Magnification, 3500 x.

FIGURE 7. Plot of the eigenvectors of the first 2 principal components based on 6 attributes of the eggs of 8 Anopheles (Anopheles) species (An. kleini, An. sinensis, An. sineroides, An. pullus, An. lindesayi japonicus, An. koreicus, An. lesteri, and An. belenrae). Attributes are defined in the Appendix.

FIGURE 8. Plot of the individual components of the eggs of 8 Anopheles (Anopheles) species (BE, An. belenrae; KL, An. kleini; KO, An. koreicus; LE, An. lesteri; LN, An. lindesayi japonicus; PU, An. pullus; SI, An. sinensis; and SR, An. sineroides).

FIGURE 9. Plot of the first 2 individual discriminant functions, based on 6 attributes of the eggs of 8 Anopheles (Anopheles) species (BE, An. belenrae; KL, An. kleini; KO, An. koreicus; LE, An. lesteri; LN, An. lindesayi japonicus; PU, An. pullus; SI, An. sinensis; and SR, An. sineroides).

FIGURE 10. Group centroids, by Anopheles (Anopheles) species, of the data plotted in Figure 9 (BE, An. belenrae; KL, An. kleini; KO, An. koreicus; LE, An. lesteri; LN, An. lindesayi japonicus; PU, An. pullus; SI, An. sinensis; and SR, An. sineroides).