**1. Introduction**

Non-target mosquito predators, including spiders and wasps, are often harmed by neurotoxic pesticides [1]. Orchards where conventional insecticides are used have far lower spider populations and species counts than unsprayed orchards [2]. Spiders with bodies larger than mosquitoes are not killed by ultra-low volume (ULV) spraying of insecticides such as permethrin [3]. However, studies such as these sometimes conflate survival with lack of harm, and they have not examined effects on small-bodied spiders, which do capture mosquitoes. Spiders exposed to sublethal pesticides change their behavior in ways that reduce prey capture. For instance, sublethal pyrethroid spray residues lower activity rates of spiders, even when the spiders contacted the pyrethroids on foliage 20 days after application [4]. Following nonlethal exposure to Spinosad, an acetylcholine disrupter, the orb-weaving spider *Agalenatea redii* showed irregularities in web design and lower prey capture activity [5]. An increase in spider migration was found within a large plantation where sublethal amounts of organophosphate pesticides were sprayed around its borders, suggesting spiders sense and actively avoid areas with some insecticide treatments [6].

Spider webs are particularly effective at capturing insecticide sprays, retaining an order of magnitude higher concentration than an equivalent area of paper [7]. Orb-weaving spiders recycle their webs daily, ingesting the spiral silk strands while leaving some of the radial structural strands intact as a scaffold for rebuilding [8–11]. By consuming the web, orb-weaving spiders ingest higher amounts of sprayed pesticides than would contact their bodies and legs. Araneidae typically construct orb webs in a vertical plane,

**Citation:** Rhoades, S.N.; Stoddard, P.K. Nonlethal Effects of Pesticides on Web-Building Spiders Might Account for Rapid Mosquito Population Rebound after Spray Application. *Appl. Sci.* **2021**, *11*, 1360. https:// doi.org/10.3390/app11041360

Academic Editor: Giuseppe Manetto Received: 17 December 2020 Accepted: 31 January 2021 Published: 3 February 2021

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whereas Tetragnathidae typically construct webs in the horizontal plane [12]. Horizontal webs should intercept more insecticide droplets following a spray application, making tetragnathids particularly vulnerable.

Following aerial insecticide application in New Orleans, yellow fever mosquitoes (*Aedes aegypti*) rebounded faster than the population model predicted [13]. The authors attributed this rapid increase to release from larval competition, a phenomenon welldocumented in the literature [14–16]. However, the data presented in that study show that the rebound occurred within the 3-day non-feeding window prior to eclosure, a period when there can be no competition for food. A similar effect could be seen in Miami's *Ae. aegypti* populations during the Zika outbreak of 2017. Adult *Ae. aegypti* in Miami's Wynwood neighborhood were virtually eliminated by aerial spraying on two occasions, only to rebound to pre-spray levels in just three days [17]. In both studies, populations leveled off after rebounding. Release from larval competition does not fit the timing window for the rapid rebound observed following aerial spraying of adulticides, but temporary release from predation might allow such a rebound to occur. Off-target effects of insecticides on mosquito predators such as spiders, even non-lethal effects, might allow greater survival of adult mosquitos during the rebound period.

Mabel's orchard orbweaver spider (*Leucauge argyrobapta,* Tetragnathidae) is found in Florida, throughout the Caribbean, and from Mexico to Brazil, with congeners common throughout the North American continent [18]. In South Florida, *L. argyrobapta* is abundant in treed areas and around residences, constructing webs in vegetation, eaves of houses, porches, and patio furnishings. Both juvenile and adult *L. argyrobapta* are capable of subduing mosquitoes. *L. argyrobapta* produces viscous horizontal orb webs, and recycles sticky spiral capture threads nightly, as is typical of tetragnathid orb-weavers [12,19].

We explored the effects of sprayed permethrin, absorbed through web contact and web recycling, on the abilities of adult *L. argyrobapta* to repair their webs and capture live adult mosquitoes. Permethrin is a type I synthetic pyrethroid used routinely to control adult mosquitoes in Miami-Dade County (FL, USA) and elsewhere. Pyrethroids bind the pore of the voltage-gated sodium channel preventing its closure, thus causing the nerves to hyperpolarize and muscle action to cease [20].

#### **2. Materials and Methods**

We chose permethrin as our insecticide because of its wide use in mosquito control in Miami-Dade County. The treatment solution was a 0.0368% solution of permethrin in acetone (1000× dilution of Martin's 36.8% Permethrin SFR, Control Solutions Inc., Pasadena, TX, USA) with a cis/trans ratio 42–58%. Two negative controls were (1) the solvent carrier (AU582) diluted 1000× in acetone, and (2) acetone alone. By trial and error, we determined that this permethrin concentration did not kill any *L. argyrobapta* when applied to their webs. To put this 0.0368% concentration in context, one hour of foot contact with 2.1% permethrin impregnated paper is the LC-50 concentration for local *Ae. aegypti*, and 0.1% is the highest concentration that kills none of them (P.K. Stoddard, unpublished). Acetone is recommended as a pyrethroid carrier in arthropod toxicity studies because it evaporates quickly [21] and is tolerated well by arthropods [22]. Because of acetone's high volatility and our limitation on number of available and comparable spiders for allocation to treatment groups, we considered acetone-only a reasonable negative control and omitted an unsprayed control group. The perfect hunting success of spiders recorded on acetone-treated webs supported our choice. At just 0.06% of the solution, the amount of AU582 carrier solvent in the sprayed permethrin solution was insignificant and had no measurable effect on spider behavior.

*L. argyrobapta* were tested near a residence within a 0.4 ha plot of native hardwood forest in Miami-Dade County (25◦3000.25", 80◦2803.48"). In March 2020, we removed 45 spiders from their webs, delicately so as not to damage the webs. We tore sections of the webs, both spiral mesh strands and several radii, to allow us to determine if the web had been recycled and reconstructed. We sprayed 15 webs with permethrin solution, 15 with acetone plus carrier, and 15 with acetone, dispensing of 2 mL of each solution per web using a hand sprayer. Webs receiving the different treatments were chosen at random with respect to the body size of the resident spider. We allowed webs to dry for 20 min then returned each spider to its original web. Webs were photographed 1 h after spraying and again 24 h later. We took two orthogonal measurements of web mesh diameter, both before spray application and again 24 h later, and noted whether the web had been substantially reconstructed (at least 75% replacement of spiral mesh).

In June 2020, we repeated the same web treatments, and determined the efficacy of treated webs and their resident spiders at retaining and subduing live mosquitoes, as well as whether the web had been substantially reconstructed. Ambient temperatures were higher in June than March, with overnight lows averaging 18 ◦C in March and 26 ◦C in June.

The mosquitoes we used were an even mix of wild *Ae. aegypti,* and *Wyeomyia vanduzeei*, captured locally with a BG-2 Sentinel trap (Biogents, Regensberg, Germany). Twenty-one webs were sprayed with permethrin and 16 with the acetone control. Using a mouth aspirator (John W. Hock Company, Gainesville, FL, USA), we aspirated five mosquitoes at random from the mosquito cage and propelled them toward an intact section of the web. Before conducting the trials, we practiced on untreated webs until we could reliably expel mosquitoes at the correct velocity to strike the web but not break the strands. Preliminary trials determined that undisturbed *L. argyrobapta* readily capture and consume any mosquito that sticks in the spiral web for at least three seconds (Figure 1). During the trials, we noted whether mosquitoes stuck to the web for 3+ s, whether the spider seized a stuck mosquito, and the latency for the spider to respond to the web strike.

**Figure 1.** (**A**) In the mosquito handling procedure, mosquitoes were propelled toward the web with a light puff of air, being careful not to blow air directly at the spider. (**B**) An adult female *L. argyrobapta* subdues a mosquito (*W. vanduzeei*) that had been propelled into the web.

Data on frequency of web repair were analyzed by comparing repaired and nonrepaired webs as categorical variables using chi-square or Fisher's Exact Test to compare the treatment groups. We calculated web area from the two diameter measurements. Diameters of reconstructed webs were identical for every spider in the two control groups but differed in the permethrin-treated group, so web areas of spiders that attempted web reconstruction were compared before and after permethrin application using a paired T-test for unequal variance. Whether at least one mosquito adhering to each web was seized by spiders in the two treatment groups was evaluated with Fisher's Exact Test. Numbers of mosquitoes captured by the two treatment groups were compared using a 2-sample *t*-test.
