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Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae)
Arthropod Structure & Development 41 (2012) 475e481
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Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae) H.C. Tuten a, *, W.C. Bridges Jr. b, P.H. Adler a a b
114 Long Hall, Entomology Program, Clemson University, Clemson, SC, USA 258 Barre Hall, Department of Mathematical Sciences, Clemson University, Clemson, SC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2011 Accepted 18 April 2012
The structure of the pyloric armature, hypothesized to aid in blood-meal digestion or parasite resistance, was compared quantitatively among the following 8 species in 5 genera of adult mosquitoes from the southeastern United States: Aedes albopictus, Aedes japonicus, Aedes triseriatus, Anopheles punctipennis, Culex pipiens s.l., Culex restuans, Orthopodomyia signifera, and Toxorhynchites rutilus. Females differed significantly among species in the structure of spines composing the armature, with Aedes spp. forming one general group, Culex spp. another, and An. punctipennis and Or. signifera a third. Relationships of species based on structural characters of the armature were consistent with recent culicid phylogenies. Although pyloric armature has been noted in mosquitoes and other insects, this is the first quantitative investigation of the mosquito pyloric armature. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Mosquito Pyloric armature Aedes Anopheles Culex
1. Introduction Chitinous spines, also called spicules or microspines, are borne on the cuticular gut lining of the cibariuim, pharynx, and pylorus of arthropods, such as the larvae of Simuliidae and Lepidoptera and the adults of Ephemeroptera, Diplopoda, and phlebotomine Psychodidae (Trembley, 1951; Byers and Bond, 1971; Christensen et al., 1971; McGreevy et al., 1978; Elzinga, 1998; Kim and Adler, 2009). The pyloric armature of adult mosquitoes is a collection of posteriorly directed spines posterior to the pyloric valve (Richins, 1938; Trembley, 1951). It has received scant attention, relative to the cibarial and pharyngeal armature. Although species differences have been reported anecdotally (Trembley, 1951; Vaughan et al., 1991), quantitative analyses are lacking. Eysell (1905) called the spines of the pyloric armature “Chitin-Nadeln” and noted they “projected downward” and were arranged in “regular” formation, possibly referring to rows. Thompson (1905) described the “ileocolon” (i.e., pylorus) as a pumping apparatus “roughened by bristlelike chitinous papillae which point caudad,” and described the armature as a “hirsute belt.” De Boissezon (1930) referred to the
* Corresponding author. Present address: Department of Entomology, University of Kentucky, S-225 Agricultural Science Center North, Lexington, KY 40546-0091, USA. Tel.: þ1 859 257 7450; fax: þ1 859 323 1120. E-mail addresses: [email protected] (H.C. Tuten), [email protected] (W.C. Bridges), [email protected] (P.H. Adler). 1467-8039/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2012.04.002
armature as “poils chitineux hérissés.” Richins (1938) described the pylorus as having “rough spines projecting caudad into the lumen,” while Snodgrass (1959) stated that “the inner wall of the pyloric funnel is armed in some species with numerous small spines directed posteriorly,” and Christophers (1960) reported “a fine cuticular lining which carries backwardly projecting spinous processes.” Christophers (1960) also noted “the spines are not unlike those seen on the larval cuticle in some situations, namely a thorn-like apex which is continued into from four to six fine spines projecting in a horizontal plane.” Trembley (1951), who presented the only light-microscope photographs, found “pyloric spines” of 6e16 mm in “irregular rows” that changed from “fine and comblike” to “heavier,” anterior to posterior, in Aedes aegypti, and reported pyloric spines in both sexes of nine additional species. Two scanning electron micrographs of the spines of Ae. aegypti were published as part of a larger study of the gut (Dapples and Lea, 1974). The pyloric armature might aid mechanical filtering and concentrating of host erythrocytes from serum, and its structure might vary with size and shape of erythrocytes (Vaughan et al., 1991; Lyimo and Ferguson, 2009). The armature, in combination with peristalsis of the pylorus, also might aid in hemolysis of host blood cells (Vaughan et al., 1991), a function attributed to the cibarial armature (Coluzzi et al., 1982; Chadee et al., 1996). The foregut armature shreds filarial nematodes (e.g., Wuchereria bancrofti) ingested in mosquito blood meals (McGreevy et al., 1978). Accordingly, the pyloric armature might aid in killing L1 larvae of Dirofilaria spp. These larvae move into the Malpighian tubules
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through openings in the pyloric valve (J. McCall, pers. comm.) e a strategy different from that of other filarioid nematodes that cross the midgut into the hemocoel (Macdonald and Ramachandran, 1965). A quantitative understanding of pyloric armature potentially can elucidate mechanisms responsible for vector competence and host choice, and aid taxonomy and phylogenetic inference. Our objective was to compare the pyloric armature of adult mosquitoes representing five genera and eight species to test the hypothesis that species differ significantly in spine structure. 2. Materials and methods 2.1. Collection and preparation Mosquitoes were obtained JuneeSeptember 2009 with gravid and light traps at the Greenville (34 50.580 N 82 23.240 W, Greenville Co.) and Riverbanks (34 00.580 N 8104.560 W, Richland Co.) zoos and AprileMay 2011 in Clemson (34 39.180 N 82 50.030 W, Pickens Co.), South Carolina. After identification, zoo samples were stored at 20 C before dissection, while Clemson samples were dissected fresh. Frozen mosquitoes were rehydrated for 1e3 days in a 10% AlconoxÒ solution in a refrigerator before dissection. Photographs and measurements were taken of the pyloric armature of four males and females each of Aedes albopictus (Skuse), five females of Aedes japonicus Yamada, two males and females each of Aedes triseriatus (Say), three females of Anopheles punctipennis (Say), four females of Culex pipiens s.l. Linnaeus, five females of Culex restuans Theobald, one female of Orthopodomyia signifera (Coquillett), and one female of Toxorhynchites rutilus (Coquillett). All images are deposited on a CD, with voucher specimens, in the Clemson University Arthropod Collection, Clemson, South Carolina. 2.2. Dissections Each mosquito was oriented laterally in a drop of phosphatebuffered saline and held with a pin through the thorax. The eighth abdominal segment was pinched with forceps, and the gut was removed by pulling the forceps posteriorly. The gut and attached eighth abdominal segment were dragged into a drop of 10% KOH and cleared for 3e4 h at room temperature, with solution added to compensate for evaporation. The gut then was dragged by the eighth abdominal segment into a drop of 50% acetic acid on the
slide. The posterior abdomen was severed from the gut and a coverslip applied to the drop with the gut, resulting in either an anterior to posterior view of the pylorus interior or a lateral view of the pylorus exterior (Fig. 1). 2.3. Terminology Because “spines” is the term most commonly used in the literature (e.g., Trembley, 1951; Vaughan et al., 1991), we use this term to describe the individual spiculate projections of the cuticular intima in the mosquito pylorus. Each spine consists of a “pedicel” and one or more “teeth.” The “base” is where the spine originates anteriorly in the pyloric intima, and the “apex” is the distal tip of each tooth. The pedicel meets the teeth at the “junction.” 2.4. Images and measurements Pyloric armature was viewed and photographed under phasecontrast at 50, 125, 250, 500, and 1250 with a Jenoptik camera (ProgRes Speed XT core 5) on an Olympus BH-2 compound microscope. The following measurements were made on photographs of the armature in the ImageJ software program (Abràmoff et al., 2004): length of the pylorus, junction width, pedicel width, pedicel length, tooth length, and number of teeth (Fig. 2). Measurements were taken for up to five spines per specimen in each of the first (anterior) and second (middle) third of the pylorus. Distances between adjacent spine bases were measured throughout the pylorus (Fig. 3). Anterior and middle spines were scored, when clearly visible, for whether 1) the line, or junction, where the teeth met the spine pedicel was straight (teeth flush) or irregular (variation in teeth attachment line); 2) the teeth were barbed (i.e., flared at the apex like a spearhead) or unbarbed; and 3) the spines were pointed and closed (i.e., base of spine coming to a complete point), pointed and open (i.e., base approaching a point but not complete), or truncate (i.e., no noticeable point) (Fig. 3). Pylori were scored for whether 1) spines in the pylorus were sparse (distance between spines > one spine width), regular (distance between spines one spine width, but not overlapping), or dense (overlapping spines); and 2) spines were or were not in horizontal rows. Posterior spines generally were less elaborate than those in the anterior and middle regions, often having only two teeth or being toothless spicules. Posterior spines, therefore, were not compared among species.
Fig. 1. Two views (phase-contrast) of slide-mounted pylori from females of Cx. pipiens s.l. Left: anterior to posterior view of the pylorus interior; right: lateral view of the pylorus exterior. Scale bar ¼ 100 um.
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2.5. Statistics
Fig. 2. Spine measurements of the pyloric armature, viewed under phase-contrast, of mosquitoes. A) Base; pedicel length: from base to line C. B) Pedicel width: of spine at midpoint of pedicel length. C) Junction width: where outside teeth meet spine pedicel. D) Tooth length: measured from C to D. Spine from female of Cx. pipiens s.l. Scale bar ¼ 5 um.
The means of spine measurements were compared among Ae. albopictus, Ae. japonicus, Ae. triseriatus, Cx. pipiens s.l., and Cx. restuans, using an analysis of variance (ANOVA) based on a model with a term for species and a term for individual within species to correct for pseudoreplication (i.e., multiple spine measurements within each individual). Before analysis, data were evaluated for conformity to ANOVA assumptions of normality and homoskedasticity. If necessary, data were square-root transformed prior to analysis. If transformation did not achieve normality and homoskedasticity, a nonparametric KruskaleWallis test was used. When only the assumption of homoskedasticity was violated, Welch’s ANOVA was used. If the results of a KruskaleWallis test or Welch’s test did not differ from those of a traditional ANOVA, the results of the ANOVA were used. If the null hypotheses of ANOVA were rejected (P < 0.05), Tukey’s Honestly Significant Difference test was used to determine which means differed significantly (P < 0.05) from one another. Variables with significantly different means were used to group the Aedes and Culex species, using a hierarchical cluster analysis with Ward’s method. Distances were calculated between all pairs of individuals, and then hierarchical clustering with Ward’s method was used to cluster individuals, using the distances. The same procedure was used to cluster species, using the means of the variables across individuals within the species to calculate distances between all pairs of species. 3. Results 3.1. Species comparisons (Tables 1e3; Fig. 4) 3.1.1. Anterior spines of females (Table 2) The mean junction widths and pedicel widths did not differ significantly among species. Mean pedicel lengths differed significantly, Ae. japonicus being significantly longer than Ae. albopictus, Cx. restuans, and Cx. pipiens s.l. Ae. japonicus and Ae. triseriatus had significantly longer teeth than did Ae. albopictus, Cx. restuans, and Cx. pipiens s.l. Ae. albopictus had significantly more teeth than did Ae. japonicus, Cx. restuans, and Cx. pipiens s.l.; Ae. triseriatus had significantly more than did Cx. restuans and Cx. pipiens s.l.; and Ae. japonicus had significantly more than did Cx. pipiens s.l. The single specimen of Tx. rutilus was not included in comparisons because of poor specimen quality; the spines in its pylorus were dense, not in obvious rows, and barbed. 3.1.2. Middle spines of females (Table 2) The mean junction widths and pedicel widths did not differ significantly among species. However, spine lengths differed significantly, with Ae. japonicus and Ae. triseriatus being longer than Cx. restuans and Cx. pipiens s.l. (Ae. albopictus did not differ from any species). Ae. japonicus and Ae. triseriatus had significantly longer teeth than did Cx. pipiens s.l. and Cx. restuans, and all four were significantly longer than Ae. albopictus. The mean number of teeth differed significantly, with Ae. albopictus having more than all other species except Ae. japonicus, and Ae. japonicus having more than both species of Culex. 3.1.3. Spine bases of females (Table 2) The mean distances between spines did not differ significantly among species
Fig. 3. Base to base spine distance represented by white line in the pyloric armature, viewed under phase-contrast, of adult mosquitoes. Spine “A” has a straight junction width, barbed teeth, and truncate base. “B” has irregular junction width, unbarbed teeth, and pointed but open base. “C” has straight junction width, barbed teeth, and closed base. Spine from female of Ae. triseriatus. Scale bar ¼ 20 um.
3.1.4. Spines of males (Table 3) Male values were not statistically compared but averages and ranges are reported
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Fig. 4. Spines of the pyloric armature in 7 species of female mosquitoes. Spines are oriented as they would be in the mosquito pylorus, with a single pedicel anteriorly and one to several teeth posteriorly. When observed in the sagittal plane, posterior regions of spines project into the pyloric lumen, while anterior regions are flush with the intima. Photographs (phase-contrast) are arranged with species in columns, and top to bottom of figure representing anterior, middle, and posterior sets of spines, respectively, in the pylorus. AeC: Or. signifera. DeF: Cx. restuans. GeI: Cx. pipiens s.l. JeL: An. punctipennis. MeO: Ae. triseriatus. PeR: Ae. japonicus. SeU: Ae. albopictus. Scale bar ¼ 10 um.
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Table 1 Numbers of spines in the anterior and middle thirds of pylori or numbers of whole pylori having given characters (actual counts presented as ratios within categories) among 7 species of mosquitoes from South Carolina, USA. Species (no. individuals examined)
No. spines
No. pylori
Anterior
Aedes albopictus female (4) Aedes albopictus male (4) Aedes japonicus (5) Aedes triseriatus female (2) Aedes triseriatus male (2) Anopheles punctipennis (3) Culex pipiens s.l. (4) Culex restuans (5) Orthopodomyia signifera (1)
Middle
Entire Pylorus
Junction line (S:NS)a
Barbed teeth (B:NB)a
Spine base (C:O:T)a
Tooth apices (S:NS)a
Barbed teeth (B:NB)a
Spine base (C:O:T)a
Pylorus spine density (Sp:Re:D)a
Spines in rows? (R:NR)a
4:16 6:4 19:4 5:5 8:2 nmb 19:1 8:16 4:1
20:0 10:5 22:0 10:0 9:1 0:9 20:0 24:0 0:5
7:7:6 13:0:7 15:3:5 3:1:6 8:0:2 10:1:2 11:8:1 10:2:12 3:1:1
2:8 6:3 15:4 9:1 4:0 nmb 13:1 7:6 nmb
10:0 8:5 19:0 9:0 3:0 nmb 14:0 13:0 nmb
6:4:0 18:0:1 17:1:1 7:0:3 4:0:0 4:1:0 13:2:0 12:1:2 nmb
0:1:3 2:0:2 0:0:5 0:1:1 0:1:1 1:2:0 0:0:4 1:0:4 0:0:1
2:2 1:3 5:0 2:0 1:1 0:3 4:0 5:0 0:1
a S:NS ¼ No. of junctions between pedicel and teeth that are either in a straight (S) or not straight (NS) line; B:NB ¼ No. of teeth with either barbed (B) or not barbed (NB) apices; C:O:T ¼ no. of spines with either pointed and closed (C), pointed and open (O), or truncate (T) bases; Sp:Re:D ¼ density of spines in pylorus either sparse (Sp), regular (Re), or dense (D); R:NR ¼ appearance of spines on pylorus either in obvious rows (R) or not (NR). b nm ¼ not measured.
3.2. Gender comparisons (Tables 2, 3) The means of the anterior and middle spines did not differ significantly (P > 0.05) between males and females of either Ae. albopictus or Ae. triseriatus for widths of junctions and pedicels, lengths of pedicels and teeth, or numbers of teeth. Females of Ae. albopictus (but not Ae. triseriatus) had significantly more teeth than did males in the anterior section of the pylorus but not in the middle section (F ¼ 6.6841; df, 1, 2; P ¼ 0.0415). 3.3. Species clustering The dendrogram resulting from cluster analysis of females grouped species within genera (Fig. 5). Cx. restuans clustered with Cx. pipiens s.l., while Ae. japonicus and Ae. triseriatus clustered more closely with one another than to Ae. albopictus 4. Discussion Significant differences exist among species in their pyloric armature. A cluster analysis based on armature characters is congruent with phylogenetic relationships presented by Harbach and Kitching (1998) and Shepard et al. (2006). Although not included in the cluster or statistical analyses, visually An. punctipennis and Or. signifera are most similar to each other, and Tx. rutilus is more similar to Aedes and Culex than to Anopheles and Orthopodomyia. Conformity between the dendrogram based on armature characters and current mosquito phylogenies suggests that minimal convergent selection is occurring. Thus, armature traits are either neutral (i.e., static after historical selection events) or maintained by currently unknown selection forces. Mosquitoes with different armature structure generally display different host affinities. The two Culex spp. are usually ornithophagic (i.e., bird feeding) and the Aedes spp. are usually mammalophagic (i.e., mammal feeding). An. punctipennis feeds on birds and mammals, and Or. signifera on amphibians, birds, and mammals. The differences in armature might relate to differences in host erythrocytes. Average erythrocyte size for mammals is 62.1 22.2 mm3, for birds 168.9 28.5 mm3, and for reptiles 398.2 121.4 mm3 (Hawkey et al., 1991), with considerable variation within classes (Wintrobe, 1933). Mosquitoes might benefit by concentrating erythrocytes from hosts with lower densities of red blood cells, an aspect that changes by an order of magnitude among
mammals (7.77 2.86 1012/l), birds (2.79 0.53 1012/l), and reptiles (0.75 0.32 1012/l) (Hawkey et al., 1991). Physiological reactions of mosquitoes to blood meals could alter properties of the peritrophic matrix (Romoser and Cody, 1975; Berner et al., 1983). If the armature plays a role in eventual breakup of the matrix, and if the matrix fluctuates with predominant host types, then some species might have more robust armature to deal with consequences to the matrix (e.g., thicker matrix). Variation in spine shape, density, and tooth number might be greater in mosquito species that switch among birds, humans, and mammals, as opposed to specializing on one host (Lyimo, 2010). Parasites in blood meals also could alter properties of the peritrophic matrix, or parasites might exert direct selection on the female pyloric armature. Of the seven hematophagous species in this study, all but Or. signifera are vectors of Dirofilaria immitis, although their vector efficiencies vary (Buxton and Mullen, 1980; Comiskey and Wesson, 1995; Watts et al., 2001), perhaps reflecting armature differences. The armature might be lethal to all filarial parasites; for example, if parasites are displaced to the posterior region of the pylorus during blood-feeding, the backward projecting spines might disrupt subsequent parasite movement to the Malpighian tubules or flight muscles. If so, pyloric spines would be expected in all mosquito species exposed to filarial parasites, and their structural differences might be correlated with differences in parasite structure (e.g., width or cuticle strength). The presence of spines in male mosquitoes and in the female of the obligately autogenous Tx. rutilus, and lack of significant differences between males and females, could be due to several factors. Further observations on larger samples and more taxa could provide insight into these factors. As suggested for sand flies, spines in males could be relics from when both males and females were putative blood-feeders (Christensen et al., 1971). Males of Ae. aegypti have been observed feeding on diuretic fluid of females (Christophers, 1960), and elaborated pyloric spines could benefit males engaging in this behavior. Neither of these hypotheses, however, accounts for the presence of robust armature in Tx. rutilus. If the pyloric armature aids in backward passage and disintegration of the peritrophic matrix (Wigglesworth, 1950), males and nonblood-feeding species, as well as blood-feeding females, might benefit, but an explanation is still needed for the variation among species. Armature also might be expected in both sexes and non-blood feeders if it aids teneral adults in the backward passage and
Species (no. spines measured)
Junction width
Pedicel width
Anteriorb Aedes albopictus (20)
4.94 0.48
3.01 0.41
Aedes japonicus (23)
4.59 0.27
2.46 0.20
Aedes triseriatus (10) Anopheles punctipennis Culex pipiens s.l. (20)
a
(9e13)
a b c
Junction width: Pedicel width ratioa
Length of teeth
No. teeth
Median no. teeth
7.97 0.41B
1.96 0.28
1.75 0.10
3.84 0.20B
7.75 0.46A
8
10.63 0.65A
2.41 0.15
1.98 0.11
6.37 0.33A
5.78 0.37B,C
6
A
A,B
A,B
5.57 0.42 2.28 0.20 3.57 0.16
3.56 0.47 1.43 0.15 2.11 0.12
9.04 0.71 10.42 1.12 6.70 0.25B
1.71 0.18 4.69 0.43 1.96 0.12
1.77 0.24 1.71 0.23 1.76 0.09
6.20 0.52 5.88 0.53 4.58 0.34B
6.90 0.59 2.67 0.53 4.10 0.29D
6.5 3 4
4.33 0.35 5.14 0.55
3.15 0.36 3.06 0.41
6.78 0.24B 9.20 0.79
1.80 0.14 1.90 0.28
1.52 0.07 1.73 0.19
4.53 0.26B 7.49 0.78
4.63 0.27C,D 7.80 0.66
4.5 8
0.45A 0.30A,B 0.31B,C 1.43 0.26C 0.36C
5.5 4 3.5 3 3 3
3.91 4.22 3.48 2.65 2.69 2.85
0.30 0.29 0.24 0.24 0.19 0.25
2.13 2.44 1.74 1.45 1.48 1.81
0.14 0.22 0.18 0.13 0.10 0.21
9.53 12.24 1.23 4.26 7.92 7.79
0.92A,B 0.59A 0.65A 1.08 0.47 B 0.26B
2.52 3.15 3.65 1.65 3.09 2.80
0.25 0.25 0.28 0.56 0.26 0.26
1.84 1.79 2.14 1.73 1.87 1.65
0.09 0.09 0.24 0.18 0.17 0.09
3.23 7.10 7.73 3.34 4.99 5.10
0.27C 0.29A 0.54A 0.01 0.29B 0.28B
5.60 4.42 3.60 2.67 2.53 2.80
Pylorus length (n)a Both Sections 213.78e244.90 (2) 392.37e462.18 (2) 308.24 (1) 127.15 (1) 195.45e240.40 (2) 191.89 (1) nm
Distance between spine bases (n)a 18.24 4.57 (13) 19.60 7.19 (124) 19.40 1.09 (63) nmc 18.15 4.75 (109) 13.65 4.25 (82) nm
Means not compared. Significantly different means within columns indicated by superscript letters (Tukey’s HSD test, P < 0.05). nm ¼ not measured.
Table 3 Means SE (mm) of spines of the armature in the anterior and middle thirds of the pylorus between 2 species of male mosquitoes from South Carolina, USA. Junction width is where the spine teeth meet the pedicel of the spine, pedicel width is the width of the spine at the middle of the pedicel length, and pedicel length is the base of the spine to the junction. Differences between males were not statistically compared. Species (no. spines measured) Anterior Aedes albopictus (5e20) Aedes triseriatus (10) Middle Aedes albopictus (9e19) Aedes triseriatus (4)
Junction width
Pedicel width
Pedicel length
Length: Junction width ratio
Junction width: Pedicel width ratio
Length of teeth
No. teeth
Median no. teeth
4.74 1.48 4.46 0.71
1.48 0.66 2.35 0.41
7.99 1.22 11.38 2.06
2.26 0.46 2.57 0.35
2.06 0.67 1.92 0.32
2.40 0.33 4.55 1.00
2.90 3.13 7.00 1.25
2 7
3.61 1.01 3.74 0.84
1.48 0.52 1.84 0.41
10.01 2.14 11.68 2.36
3.07 1.42 3.19 0.68
2.15 0.68 2.07 0.53
3.55 1.11 5.14 0.83
2.16 2.69 5.75 1.26
0 6
Pylorus length (no. pylori measured) Both Sections 273.38 190.82 (4) 152.88 (1)
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Culex restuans (24) Orthopodomyia signifera a (5) Middleb Aedes albopictus (10) Aedes japonicus (19) Aedes triseriatus (10) Anopheles punctipennis a (2e5) Culex pipiens (14e15) Culex restuans (13e15)
Pedicel length: Junction width ratioa
Pedicel length
480
Table 2 Means SE (mm) of spines of the pyloric armature in the anterior and middle thirds of the pylorus among 7 species of female mosquitoes from South Carolina, USA. Junction width is where the spine teeth meet the pedicel of the spine, pedicel width is the width of the spine at the middle of the pedicel length, and pedicel length is the base of the spine to the junction.
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Fig. 5. Dendrogram of 5 species of mosquitoes from South Carolina, USA, based on overall spine measurements in pyloric armature of females.
egestion of the meconial peritrophic matrices (MPM1 and MPM2), which vary in occurrence and persistence among mosquito species (Romoser et al., 2000; Moll et al., 2001). The putative function of these matrices is bacterial sequestration in the pupal midgut prior to adult eclosion (MPM1) and sequestration of MPM1 and molting fluid in the pupa and teneral adult (MPM2) (Romoser et al., 2000; Moll et al., 2001). If the evolved function of the pyloric armature is to aid backward passage and disintegration of the meconial peritrophic matrix, the armature might have been exapted by female mosquitoes to aid in blood-meal processing or concentration or to protect against parasites. Pharyngeal armature occurs in both male and female mosquitoes but has been implicated in damage to ingested microfilariae (McGreevy et al., 1978). We have shown that significant variation exists in characters of the pyloric armature of mosquitoes and that armature characters correspond with phylogenetic relationships. Although we propose hypotheses to explain structural variation in pyloric armature of males and females among taxa, the functional significance of the armature remains unclear. Females, but not males, of some species of corethrellids have pyloric armature, and females of a bloodfeeding ceratopogonid in the Culicoides piliferus group do not have visible pyloric armature (Tuten, 2011). The lack of a clear pattern in the presence of pyloric armature across gender and species suggests that the armature serves multiple functions.
Acknowledgments The authors thank the staff of both zoos for facilitating mosquito collections and J. McCall (University of Georgia) for sharing his knowledge of D. immitis. This is Technical Contribution No. 5996 of the Clemson University Experiment Station, and is based on work supported, in part, by NIFA/USDA, under project number SC1700276.
References Abràmoff, M.D., Magalhães, P.J., Ram, S.J., 2004. Image processing with ImageJ. BioPhotonics International 11, 36e42. Berner, R., Rudin, W., Hecker, H., 1983. Peritrophic matrixes and protease activity in the midgut of the malaria mosquito, Anopheles stephensi (Liston) (Insecta: Diptera) under normal and experimental conditions. Journal of Ultrastructure Research 83, 195e204. Buxton, B.A., Mullen, G.R., 1980. Field isolations of Dirofilaria from mosquitoes in Alabama. Journal of Parasitology 66, 140e144. Byers, J.R., Bond, E.F., 1971. Surface specializations of the hindgut cuticle of lepidopterous larvae. Canadian Journal of Zoology 49, 867e876. Chadee, D.D., Beier, J.C., Martinez, R., 1996. The effect of the cibarial armature on blood meal haemolysis of four anopheline mosquitoes. Bulletin of Entomological Research 86, 351e354. Christensen, H.A., Herrer, A., Fairchild, G.B., 1971. Pyloric armature of New World phlebotomine sandflies (Diptera, Psychodidae). Journal of Medical Entomology 8, 116e119. Christophers, S., 1960. Aëdes aegypti (L.) the Yellow Fever Mosquito: Its Life History, Bionomics and Structure. Syndics of Cambridge Univ. Press, London.
Coluzzi, M., Concetti, A., Ascoli, F., 1982. Effect of cibarial armature of mosquitoes (Diptera, Culicidae) on blood-meal haemolysis. Journal of Insect Physiology 28, 885e888. Comiskey, N., Wesson, D.M., 1995. Dirofilaria (Filarioidea: Onchocercidae) infection in Aedes albopictus (Diptera: Culicidae) collected in Louisiana. Journal of Medical Entomology 32, 734e737. Dapples, C.C., Lea, A.O., 1974. Inner surface morphology of the alimentary canal in Aedes aegypti (L.) (Diptera: Culicidae). International Journal of Insect Morphology and Embryology 3, 433e442. de Boissezon, P., 1930. Contribution à l’étude de la biologie et de l’histophysiologie de Culex pipiens L. Archives of Zoological Experimental Genetics 70, 281e431. Elzinga, R.J., 1998. Microspines in the alimentary canal of Arthropoda, Onychophora, Annelida. International Journal of Insect Morphology and Embryology 27, 341e349. Eysell, A., 1905. Die Stechmücken. In: Mense, C. (Ed.). Handbuch der Tropenkrankheiten vol. II, J. A. Barth, Leipzig, pp. 97e183. Harbach, R.E., Kitching, I.J., 1998. Phylogeny and classification of the Culicidae (Diptera). Systematic Entomology 23, 327e370. Hawkey, C.M., Bennett, P.M., Gascoyne, S.C., Hart, M.G., Kirkwood, J.K., 1991. Erythrocyte size, number and haemoglobin content in vertebrates. British Journal of Haematology 77, 392e397. Kim, S.K., Adler, P.H., 2009. Digestive system of larval black flies (Diptera: Simuliidae): structure and phylogeny. Invertebrate Biology 128, 182e204. Lyimo, I.N., Ferguson, H.M., 2009. Ecological and evolutionary determinants of host species choice in mosquito vectors. Trends in Parasitology 25, 189e196. Lyimo, I.N., 2010. Ecological and evolutionary determinants of anopheline host species choice and its implications for malaria transmission. Ph. D. Thesis. Univ. Glasgow, UK. 222 pp. Macdonald, W.W., Ramachandran, C.P., 1965. The influence of the gene fm (filarial susceptibility, Brugia malayi) on the susceptibility of Aedes aegypti to seven strains of Brugia, Wuchereria and Dirofilaria. Annals of Tropical Medicine and Parasitology 59, 64e73. McGreevy, P.B., Bryan, J.H., Oothuman, P., Kolstrup, N., 1978. The lethal effects of the cibarial and pharyngeal armature of mosquitoes on microfilariae. Transactions of the Royal Society of Tropical Medicine and Hygiene 72, 361e368. Moll, R.M., Romoser, W.S., Modrakowski, M.C., Moncayo, A.C., Lerdthusnee, K., 2001. Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. Journal of Medical Entomology 38, 29e32. Richins, C.A., 1938. The metamorphosis of the digestive tract of Aedes dorsalis Meigen. Annals of the Entomological Society of America 31, 69e73. Romoser, W.S., Cody, E., 1975. The formation and fate of the peritrophic matrix in adult Culex nigripalpus (Diptera: Culicidae). Journal of Medical Entomology 12, 371e378. Romoser, W.S., Moll, R.M., Moncayo, A.C., Lerdthusnee, K., 2000. The occurrence and fate of the meconium and meconial peritrophic matrixes in pupal and adult mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 37, 893e896. Shepard, J.J., Andreadis, T.G., Vossbrinck, C.R., 2006. Molecular phylogeny and evolutionary relationships among mosquitoes (Diptera: Culicidae) from the northeastern United States based on small subunit ribosomal DNA (18s rDNA) sequences. Journal of Medical Entomology 43, 443e454. Snodgrass, R.E., 1959. The Anatomical Life of the Mosquito. Smithsonian Institution, Washington DC. 61pp. Thompson, M.T., 1905. Alimentary canal of the mosquito. Proceedings of the Boston Society of Natural History 32, 145e202. Trembley, H.L., 1951. Pyloric spines in mosquitoes. Journal of the National Malaria Society 10, 213e215. Tuten, H.C., 2011. Zoos as experiment environments: biology of larval and adult mosquitoes (Diptera: Culicidae). Ph. D. Dissertation. Clemson University, SC, USA. 173pp. Vaughan, J.A., Noden, B.H., Beier, J.C., 1991. Concentration of human erythrocytes by anopheline mosquitoes (Diptera: Culicidae) during feeding. Journal of Medical Entomology 28, 780e786. Watts, K.J., Reddy, G.R., Holmes, R.A., Lok, J.B., Knight, D.H., Smith, G., Courtney, C.H., 2001. Seasonal prevalence of third-stage larvae of Dirofilaria immitis in mosquitoes from Florida and Louisiana. Journal of Parasitology 87, 322e329. Wigglesworth, V.B., 1950. The Principles of Insect Physiology, forth ed. Methuen & Co. Ltd., London. Wintrobe, M.M., 1933. Variations in the size and haemoglobin content in erythrocytes in the blood of various vertebrates. Folia Haematologica 51, 32e49.
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Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd
Comparative morphology of the pyloric armature of adult mosquitoes (Diptera: Culicidae) H.C. Tuten a, *, W.C. Bridges Jr. b, P.H. Adler a a b
114 Long Hall, Entomology Program, Clemson University, Clemson, SC, USA 258 Barre Hall, Department of Mathematical Sciences, Clemson University, Clemson, SC, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 December 2011 Accepted 18 April 2012
The structure of the pyloric armature, hypothesized to aid in blood-meal digestion or parasite resistance, was compared quantitatively among the following 8 species in 5 genera of adult mosquitoes from the southeastern United States: Aedes albopictus, Aedes japonicus, Aedes triseriatus, Anopheles punctipennis, Culex pipiens s.l., Culex restuans, Orthopodomyia signifera, and Toxorhynchites rutilus. Females differed significantly among species in the structure of spines composing the armature, with Aedes spp. forming one general group, Culex spp. another, and An. punctipennis and Or. signifera a third. Relationships of species based on structural characters of the armature were consistent with recent culicid phylogenies. Although pyloric armature has been noted in mosquitoes and other insects, this is the first quantitative investigation of the mosquito pyloric armature. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Mosquito Pyloric armature Aedes Anopheles Culex
1. Introduction Chitinous spines, also called spicules or microspines, are borne on the cuticular gut lining of the cibariuim, pharynx, and pylorus of arthropods, such as the larvae of Simuliidae and Lepidoptera and the adults of Ephemeroptera, Diplopoda, and phlebotomine Psychodidae (Trembley, 1951; Byers and Bond, 1971; Christensen et al., 1971; McGreevy et al., 1978; Elzinga, 1998; Kim and Adler, 2009). The pyloric armature of adult mosquitoes is a collection of posteriorly directed spines posterior to the pyloric valve (Richins, 1938; Trembley, 1951). It has received scant attention, relative to the cibarial and pharyngeal armature. Although species differences have been reported anecdotally (Trembley, 1951; Vaughan et al., 1991), quantitative analyses are lacking. Eysell (1905) called the spines of the pyloric armature “Chitin-Nadeln” and noted they “projected downward” and were arranged in “regular” formation, possibly referring to rows. Thompson (1905) described the “ileocolon” (i.e., pylorus) as a pumping apparatus “roughened by bristlelike chitinous papillae which point caudad,” and described the armature as a “hirsute belt.” De Boissezon (1930) referred to the
* Corresponding author. Present address: Department of Entomology, University of Kentucky, S-225 Agricultural Science Center North, Lexington, KY 40546-0091, USA. Tel.: þ1 859 257 7450; fax: þ1 859 323 1120. E-mail addresses: [email protected] (H.C. Tuten), [email protected] (W.C. Bridges), [email protected] (P.H. Adler). 1467-8039/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2012.04.002
armature as “poils chitineux hérissés.” Richins (1938) described the pylorus as having “rough spines projecting caudad into the lumen,” while Snodgrass (1959) stated that “the inner wall of the pyloric funnel is armed in some species with numerous small spines directed posteriorly,” and Christophers (1960) reported “a fine cuticular lining which carries backwardly projecting spinous processes.” Christophers (1960) also noted “the spines are not unlike those seen on the larval cuticle in some situations, namely a thorn-like apex which is continued into from four to six fine spines projecting in a horizontal plane.” Trembley (1951), who presented the only light-microscope photographs, found “pyloric spines” of 6e16 mm in “irregular rows” that changed from “fine and comblike” to “heavier,” anterior to posterior, in Aedes aegypti, and reported pyloric spines in both sexes of nine additional species. Two scanning electron micrographs of the spines of Ae. aegypti were published as part of a larger study of the gut (Dapples and Lea, 1974). The pyloric armature might aid mechanical filtering and concentrating of host erythrocytes from serum, and its structure might vary with size and shape of erythrocytes (Vaughan et al., 1991; Lyimo and Ferguson, 2009). The armature, in combination with peristalsis of the pylorus, also might aid in hemolysis of host blood cells (Vaughan et al., 1991), a function attributed to the cibarial armature (Coluzzi et al., 1982; Chadee et al., 1996). The foregut armature shreds filarial nematodes (e.g., Wuchereria bancrofti) ingested in mosquito blood meals (McGreevy et al., 1978). Accordingly, the pyloric armature might aid in killing L1 larvae of Dirofilaria spp. These larvae move into the Malpighian tubules
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through openings in the pyloric valve (J. McCall, pers. comm.) e a strategy different from that of other filarioid nematodes that cross the midgut into the hemocoel (Macdonald and Ramachandran, 1965). A quantitative understanding of pyloric armature potentially can elucidate mechanisms responsible for vector competence and host choice, and aid taxonomy and phylogenetic inference. Our objective was to compare the pyloric armature of adult mosquitoes representing five genera and eight species to test the hypothesis that species differ significantly in spine structure. 2. Materials and methods 2.1. Collection and preparation Mosquitoes were obtained JuneeSeptember 2009 with gravid and light traps at the Greenville (34 50.580 N 82 23.240 W, Greenville Co.) and Riverbanks (34 00.580 N 8104.560 W, Richland Co.) zoos and AprileMay 2011 in Clemson (34 39.180 N 82 50.030 W, Pickens Co.), South Carolina. After identification, zoo samples were stored at 20 C before dissection, while Clemson samples were dissected fresh. Frozen mosquitoes were rehydrated for 1e3 days in a 10% AlconoxÒ solution in a refrigerator before dissection. Photographs and measurements were taken of the pyloric armature of four males and females each of Aedes albopictus (Skuse), five females of Aedes japonicus Yamada, two males and females each of Aedes triseriatus (Say), three females of Anopheles punctipennis (Say), four females of Culex pipiens s.l. Linnaeus, five females of Culex restuans Theobald, one female of Orthopodomyia signifera (Coquillett), and one female of Toxorhynchites rutilus (Coquillett). All images are deposited on a CD, with voucher specimens, in the Clemson University Arthropod Collection, Clemson, South Carolina. 2.2. Dissections Each mosquito was oriented laterally in a drop of phosphatebuffered saline and held with a pin through the thorax. The eighth abdominal segment was pinched with forceps, and the gut was removed by pulling the forceps posteriorly. The gut and attached eighth abdominal segment were dragged into a drop of 10% KOH and cleared for 3e4 h at room temperature, with solution added to compensate for evaporation. The gut then was dragged by the eighth abdominal segment into a drop of 50% acetic acid on the
slide. The posterior abdomen was severed from the gut and a coverslip applied to the drop with the gut, resulting in either an anterior to posterior view of the pylorus interior or a lateral view of the pylorus exterior (Fig. 1). 2.3. Terminology Because “spines” is the term most commonly used in the literature (e.g., Trembley, 1951; Vaughan et al., 1991), we use this term to describe the individual spiculate projections of the cuticular intima in the mosquito pylorus. Each spine consists of a “pedicel” and one or more “teeth.” The “base” is where the spine originates anteriorly in the pyloric intima, and the “apex” is the distal tip of each tooth. The pedicel meets the teeth at the “junction.” 2.4. Images and measurements Pyloric armature was viewed and photographed under phasecontrast at 50, 125, 250, 500, and 1250 with a Jenoptik camera (ProgRes Speed XT core 5) on an Olympus BH-2 compound microscope. The following measurements were made on photographs of the armature in the ImageJ software program (Abràmoff et al., 2004): length of the pylorus, junction width, pedicel width, pedicel length, tooth length, and number of teeth (Fig. 2). Measurements were taken for up to five spines per specimen in each of the first (anterior) and second (middle) third of the pylorus. Distances between adjacent spine bases were measured throughout the pylorus (Fig. 3). Anterior and middle spines were scored, when clearly visible, for whether 1) the line, or junction, where the teeth met the spine pedicel was straight (teeth flush) or irregular (variation in teeth attachment line); 2) the teeth were barbed (i.e., flared at the apex like a spearhead) or unbarbed; and 3) the spines were pointed and closed (i.e., base of spine coming to a complete point), pointed and open (i.e., base approaching a point but not complete), or truncate (i.e., no noticeable point) (Fig. 3). Pylori were scored for whether 1) spines in the pylorus were sparse (distance between spines > one spine width), regular (distance between spines one spine width, but not overlapping), or dense (overlapping spines); and 2) spines were or were not in horizontal rows. Posterior spines generally were less elaborate than those in the anterior and middle regions, often having only two teeth or being toothless spicules. Posterior spines, therefore, were not compared among species.
Fig. 1. Two views (phase-contrast) of slide-mounted pylori from females of Cx. pipiens s.l. Left: anterior to posterior view of the pylorus interior; right: lateral view of the pylorus exterior. Scale bar ¼ 100 um.
H.C. Tuten et al. / Arthropod Structure & Development 41 (2012) 475e481
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2.5. Statistics
Fig. 2. Spine measurements of the pyloric armature, viewed under phase-contrast, of mosquitoes. A) Base; pedicel length: from base to line C. B) Pedicel width: of spine at midpoint of pedicel length. C) Junction width: where outside teeth meet spine pedicel. D) Tooth length: measured from C to D. Spine from female of Cx. pipiens s.l. Scale bar ¼ 5 um.
The means of spine measurements were compared among Ae. albopictus, Ae. japonicus, Ae. triseriatus, Cx. pipiens s.l., and Cx. restuans, using an analysis of variance (ANOVA) based on a model with a term for species and a term for individual within species to correct for pseudoreplication (i.e., multiple spine measurements within each individual). Before analysis, data were evaluated for conformity to ANOVA assumptions of normality and homoskedasticity. If necessary, data were square-root transformed prior to analysis. If transformation did not achieve normality and homoskedasticity, a nonparametric KruskaleWallis test was used. When only the assumption of homoskedasticity was violated, Welch’s ANOVA was used. If the results of a KruskaleWallis test or Welch’s test did not differ from those of a traditional ANOVA, the results of the ANOVA were used. If the null hypotheses of ANOVA were rejected (P < 0.05), Tukey’s Honestly Significant Difference test was used to determine which means differed significantly (P < 0.05) from one another. Variables with significantly different means were used to group the Aedes and Culex species, using a hierarchical cluster analysis with Ward’s method. Distances were calculated between all pairs of individuals, and then hierarchical clustering with Ward’s method was used to cluster individuals, using the distances. The same procedure was used to cluster species, using the means of the variables across individuals within the species to calculate distances between all pairs of species. 3. Results 3.1. Species comparisons (Tables 1e3; Fig. 4) 3.1.1. Anterior spines of females (Table 2) The mean junction widths and pedicel widths did not differ significantly among species. Mean pedicel lengths differed significantly, Ae. japonicus being significantly longer than Ae. albopictus, Cx. restuans, and Cx. pipiens s.l. Ae. japonicus and Ae. triseriatus had significantly longer teeth than did Ae. albopictus, Cx. restuans, and Cx. pipiens s.l. Ae. albopictus had significantly more teeth than did Ae. japonicus, Cx. restuans, and Cx. pipiens s.l.; Ae. triseriatus had significantly more than did Cx. restuans and Cx. pipiens s.l.; and Ae. japonicus had significantly more than did Cx. pipiens s.l. The single specimen of Tx. rutilus was not included in comparisons because of poor specimen quality; the spines in its pylorus were dense, not in obvious rows, and barbed. 3.1.2. Middle spines of females (Table 2) The mean junction widths and pedicel widths did not differ significantly among species. However, spine lengths differed significantly, with Ae. japonicus and Ae. triseriatus being longer than Cx. restuans and Cx. pipiens s.l. (Ae. albopictus did not differ from any species). Ae. japonicus and Ae. triseriatus had significantly longer teeth than did Cx. pipiens s.l. and Cx. restuans, and all four were significantly longer than Ae. albopictus. The mean number of teeth differed significantly, with Ae. albopictus having more than all other species except Ae. japonicus, and Ae. japonicus having more than both species of Culex. 3.1.3. Spine bases of females (Table 2) The mean distances between spines did not differ significantly among species
Fig. 3. Base to base spine distance represented by white line in the pyloric armature, viewed under phase-contrast, of adult mosquitoes. Spine “A” has a straight junction width, barbed teeth, and truncate base. “B” has irregular junction width, unbarbed teeth, and pointed but open base. “C” has straight junction width, barbed teeth, and closed base. Spine from female of Ae. triseriatus. Scale bar ¼ 20 um.
3.1.4. Spines of males (Table 3) Male values were not statistically compared but averages and ranges are reported
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Fig. 4. Spines of the pyloric armature in 7 species of female mosquitoes. Spines are oriented as they would be in the mosquito pylorus, with a single pedicel anteriorly and one to several teeth posteriorly. When observed in the sagittal plane, posterior regions of spines project into the pyloric lumen, while anterior regions are flush with the intima. Photographs (phase-contrast) are arranged with species in columns, and top to bottom of figure representing anterior, middle, and posterior sets of spines, respectively, in the pylorus. AeC: Or. signifera. DeF: Cx. restuans. GeI: Cx. pipiens s.l. JeL: An. punctipennis. MeO: Ae. triseriatus. PeR: Ae. japonicus. SeU: Ae. albopictus. Scale bar ¼ 10 um.
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Table 1 Numbers of spines in the anterior and middle thirds of pylori or numbers of whole pylori having given characters (actual counts presented as ratios within categories) among 7 species of mosquitoes from South Carolina, USA. Species (no. individuals examined)
No. spines
No. pylori
Anterior
Aedes albopictus female (4) Aedes albopictus male (4) Aedes japonicus (5) Aedes triseriatus female (2) Aedes triseriatus male (2) Anopheles punctipennis (3) Culex pipiens s.l. (4) Culex restuans (5) Orthopodomyia signifera (1)
Middle
Entire Pylorus
Junction line (S:NS)a
Barbed teeth (B:NB)a
Spine base (C:O:T)a
Tooth apices (S:NS)a
Barbed teeth (B:NB)a
Spine base (C:O:T)a
Pylorus spine density (Sp:Re:D)a
Spines in rows? (R:NR)a
4:16 6:4 19:4 5:5 8:2 nmb 19:1 8:16 4:1
20:0 10:5 22:0 10:0 9:1 0:9 20:0 24:0 0:5
7:7:6 13:0:7 15:3:5 3:1:6 8:0:2 10:1:2 11:8:1 10:2:12 3:1:1
2:8 6:3 15:4 9:1 4:0 nmb 13:1 7:6 nmb
10:0 8:5 19:0 9:0 3:0 nmb 14:0 13:0 nmb
6:4:0 18:0:1 17:1:1 7:0:3 4:0:0 4:1:0 13:2:0 12:1:2 nmb
0:1:3 2:0:2 0:0:5 0:1:1 0:1:1 1:2:0 0:0:4 1:0:4 0:0:1
2:2 1:3 5:0 2:0 1:1 0:3 4:0 5:0 0:1
a S:NS ¼ No. of junctions between pedicel and teeth that are either in a straight (S) or not straight (NS) line; B:NB ¼ No. of teeth with either barbed (B) or not barbed (NB) apices; C:O:T ¼ no. of spines with either pointed and closed (C), pointed and open (O), or truncate (T) bases; Sp:Re:D ¼ density of spines in pylorus either sparse (Sp), regular (Re), or dense (D); R:NR ¼ appearance of spines on pylorus either in obvious rows (R) or not (NR). b nm ¼ not measured.
3.2. Gender comparisons (Tables 2, 3) The means of the anterior and middle spines did not differ significantly (P > 0.05) between males and females of either Ae. albopictus or Ae. triseriatus for widths of junctions and pedicels, lengths of pedicels and teeth, or numbers of teeth. Females of Ae. albopictus (but not Ae. triseriatus) had significantly more teeth than did males in the anterior section of the pylorus but not in the middle section (F ¼ 6.6841; df, 1, 2; P ¼ 0.0415). 3.3. Species clustering The dendrogram resulting from cluster analysis of females grouped species within genera (Fig. 5). Cx. restuans clustered with Cx. pipiens s.l., while Ae. japonicus and Ae. triseriatus clustered more closely with one another than to Ae. albopictus 4. Discussion Significant differences exist among species in their pyloric armature. A cluster analysis based on armature characters is congruent with phylogenetic relationships presented by Harbach and Kitching (1998) and Shepard et al. (2006). Although not included in the cluster or statistical analyses, visually An. punctipennis and Or. signifera are most similar to each other, and Tx. rutilus is more similar to Aedes and Culex than to Anopheles and Orthopodomyia. Conformity between the dendrogram based on armature characters and current mosquito phylogenies suggests that minimal convergent selection is occurring. Thus, armature traits are either neutral (i.e., static after historical selection events) or maintained by currently unknown selection forces. Mosquitoes with different armature structure generally display different host affinities. The two Culex spp. are usually ornithophagic (i.e., bird feeding) and the Aedes spp. are usually mammalophagic (i.e., mammal feeding). An. punctipennis feeds on birds and mammals, and Or. signifera on amphibians, birds, and mammals. The differences in armature might relate to differences in host erythrocytes. Average erythrocyte size for mammals is 62.1 22.2 mm3, for birds 168.9 28.5 mm3, and for reptiles 398.2 121.4 mm3 (Hawkey et al., 1991), with considerable variation within classes (Wintrobe, 1933). Mosquitoes might benefit by concentrating erythrocytes from hosts with lower densities of red blood cells, an aspect that changes by an order of magnitude among
mammals (7.77 2.86 1012/l), birds (2.79 0.53 1012/l), and reptiles (0.75 0.32 1012/l) (Hawkey et al., 1991). Physiological reactions of mosquitoes to blood meals could alter properties of the peritrophic matrix (Romoser and Cody, 1975; Berner et al., 1983). If the armature plays a role in eventual breakup of the matrix, and if the matrix fluctuates with predominant host types, then some species might have more robust armature to deal with consequences to the matrix (e.g., thicker matrix). Variation in spine shape, density, and tooth number might be greater in mosquito species that switch among birds, humans, and mammals, as opposed to specializing on one host (Lyimo, 2010). Parasites in blood meals also could alter properties of the peritrophic matrix, or parasites might exert direct selection on the female pyloric armature. Of the seven hematophagous species in this study, all but Or. signifera are vectors of Dirofilaria immitis, although their vector efficiencies vary (Buxton and Mullen, 1980; Comiskey and Wesson, 1995; Watts et al., 2001), perhaps reflecting armature differences. The armature might be lethal to all filarial parasites; for example, if parasites are displaced to the posterior region of the pylorus during blood-feeding, the backward projecting spines might disrupt subsequent parasite movement to the Malpighian tubules or flight muscles. If so, pyloric spines would be expected in all mosquito species exposed to filarial parasites, and their structural differences might be correlated with differences in parasite structure (e.g., width or cuticle strength). The presence of spines in male mosquitoes and in the female of the obligately autogenous Tx. rutilus, and lack of significant differences between males and females, could be due to several factors. Further observations on larger samples and more taxa could provide insight into these factors. As suggested for sand flies, spines in males could be relics from when both males and females were putative blood-feeders (Christensen et al., 1971). Males of Ae. aegypti have been observed feeding on diuretic fluid of females (Christophers, 1960), and elaborated pyloric spines could benefit males engaging in this behavior. Neither of these hypotheses, however, accounts for the presence of robust armature in Tx. rutilus. If the pyloric armature aids in backward passage and disintegration of the peritrophic matrix (Wigglesworth, 1950), males and nonblood-feeding species, as well as blood-feeding females, might benefit, but an explanation is still needed for the variation among species. Armature also might be expected in both sexes and non-blood feeders if it aids teneral adults in the backward passage and
Species (no. spines measured)
Junction width
Pedicel width
Anteriorb Aedes albopictus (20)
4.94 0.48
3.01 0.41
Aedes japonicus (23)
4.59 0.27
2.46 0.20
Aedes triseriatus (10) Anopheles punctipennis Culex pipiens s.l. (20)
a
(9e13)
a b c
Junction width: Pedicel width ratioa
Length of teeth
No. teeth
Median no. teeth
7.97 0.41B
1.96 0.28
1.75 0.10
3.84 0.20B
7.75 0.46A
8
10.63 0.65A
2.41 0.15
1.98 0.11
6.37 0.33A
5.78 0.37B,C
6
A
A,B
A,B
5.57 0.42 2.28 0.20 3.57 0.16
3.56 0.47 1.43 0.15 2.11 0.12
9.04 0.71 10.42 1.12 6.70 0.25B
1.71 0.18 4.69 0.43 1.96 0.12
1.77 0.24 1.71 0.23 1.76 0.09
6.20 0.52 5.88 0.53 4.58 0.34B
6.90 0.59 2.67 0.53 4.10 0.29D
6.5 3 4
4.33 0.35 5.14 0.55
3.15 0.36 3.06 0.41
6.78 0.24B 9.20 0.79
1.80 0.14 1.90 0.28
1.52 0.07 1.73 0.19
4.53 0.26B 7.49 0.78
4.63 0.27C,D 7.80 0.66
4.5 8
0.45A 0.30A,B 0.31B,C 1.43 0.26C 0.36C
5.5 4 3.5 3 3 3
3.91 4.22 3.48 2.65 2.69 2.85
0.30 0.29 0.24 0.24 0.19 0.25
2.13 2.44 1.74 1.45 1.48 1.81
0.14 0.22 0.18 0.13 0.10 0.21
9.53 12.24 1.23 4.26 7.92 7.79
0.92A,B 0.59A 0.65A 1.08 0.47 B 0.26B
2.52 3.15 3.65 1.65 3.09 2.80
0.25 0.25 0.28 0.56 0.26 0.26
1.84 1.79 2.14 1.73 1.87 1.65
0.09 0.09 0.24 0.18 0.17 0.09
3.23 7.10 7.73 3.34 4.99 5.10
0.27C 0.29A 0.54A 0.01 0.29B 0.28B
5.60 4.42 3.60 2.67 2.53 2.80
Pylorus length (n)a Both Sections 213.78e244.90 (2) 392.37e462.18 (2) 308.24 (1) 127.15 (1) 195.45e240.40 (2) 191.89 (1) nm
Distance between spine bases (n)a 18.24 4.57 (13) 19.60 7.19 (124) 19.40 1.09 (63) nmc 18.15 4.75 (109) 13.65 4.25 (82) nm
Means not compared. Significantly different means within columns indicated by superscript letters (Tukey’s HSD test, P < 0.05). nm ¼ not measured.
Table 3 Means SE (mm) of spines of the armature in the anterior and middle thirds of the pylorus between 2 species of male mosquitoes from South Carolina, USA. Junction width is where the spine teeth meet the pedicel of the spine, pedicel width is the width of the spine at the middle of the pedicel length, and pedicel length is the base of the spine to the junction. Differences between males were not statistically compared. Species (no. spines measured) Anterior Aedes albopictus (5e20) Aedes triseriatus (10) Middle Aedes albopictus (9e19) Aedes triseriatus (4)
Junction width
Pedicel width
Pedicel length
Length: Junction width ratio
Junction width: Pedicel width ratio
Length of teeth
No. teeth
Median no. teeth
4.74 1.48 4.46 0.71
1.48 0.66 2.35 0.41
7.99 1.22 11.38 2.06
2.26 0.46 2.57 0.35
2.06 0.67 1.92 0.32
2.40 0.33 4.55 1.00
2.90 3.13 7.00 1.25
2 7
3.61 1.01 3.74 0.84
1.48 0.52 1.84 0.41
10.01 2.14 11.68 2.36
3.07 1.42 3.19 0.68
2.15 0.68 2.07 0.53
3.55 1.11 5.14 0.83
2.16 2.69 5.75 1.26
0 6
Pylorus length (no. pylori measured) Both Sections 273.38 190.82 (4) 152.88 (1)
H.C. Tuten et al. / Arthropod Structure & Development 41 (2012) 475e481
Culex restuans (24) Orthopodomyia signifera a (5) Middleb Aedes albopictus (10) Aedes japonicus (19) Aedes triseriatus (10) Anopheles punctipennis a (2e5) Culex pipiens (14e15) Culex restuans (13e15)
Pedicel length: Junction width ratioa
Pedicel length
480
Table 2 Means SE (mm) of spines of the pyloric armature in the anterior and middle thirds of the pylorus among 7 species of female mosquitoes from South Carolina, USA. Junction width is where the spine teeth meet the pedicel of the spine, pedicel width is the width of the spine at the middle of the pedicel length, and pedicel length is the base of the spine to the junction.
H.C. Tuten et al. / Arthropod Structure & Development 41 (2012) 475e481
481
Fig. 5. Dendrogram of 5 species of mosquitoes from South Carolina, USA, based on overall spine measurements in pyloric armature of females.
egestion of the meconial peritrophic matrices (MPM1 and MPM2), which vary in occurrence and persistence among mosquito species (Romoser et al., 2000; Moll et al., 2001). The putative function of these matrices is bacterial sequestration in the pupal midgut prior to adult eclosion (MPM1) and sequestration of MPM1 and molting fluid in the pupa and teneral adult (MPM2) (Romoser et al., 2000; Moll et al., 2001). If the evolved function of the pyloric armature is to aid backward passage and disintegration of the meconial peritrophic matrix, the armature might have been exapted by female mosquitoes to aid in blood-meal processing or concentration or to protect against parasites. Pharyngeal armature occurs in both male and female mosquitoes but has been implicated in damage to ingested microfilariae (McGreevy et al., 1978). We have shown that significant variation exists in characters of the pyloric armature of mosquitoes and that armature characters correspond with phylogenetic relationships. Although we propose hypotheses to explain structural variation in pyloric armature of males and females among taxa, the functional significance of the armature remains unclear. Females, but not males, of some species of corethrellids have pyloric armature, and females of a bloodfeeding ceratopogonid in the Culicoides piliferus group do not have visible pyloric armature (Tuten, 2011). The lack of a clear pattern in the presence of pyloric armature across gender and species suggests that the armature serves multiple functions.
Acknowledgments The authors thank the staff of both zoos for facilitating mosquito collections and J. McCall (University of Georgia) for sharing his knowledge of D. immitis. This is Technical Contribution No. 5996 of the Clemson University Experiment Station, and is based on work supported, in part, by NIFA/USDA, under project number SC1700276.
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